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RETURN  THIS  BOOK 
TO  THE  LIBRARY  OF 
DR.  STAFFORD  McLEAN 


WORKS  OF 
PROFESSOR    J.    A.    MANDEL 

PUBLISHED   BY 

JOHN  WILEY  &  SONS. 


Hand-book  for  Bio-chemical  Laboratory. 

i2mo,  cloth,  $1.50. 

TR  A  NSL  A  TIONS. 
A  Text-book  of  Physiological  Chemistry. 

By  Olof  Hammarsten,  Professor  of  Medical  and  Physio- 
logical Chemistry  in  the  University  of  Upsala.  Author- 
ized translation,  from  the  author's  enlarged  and  revised 
5th  German  edition,  by  John  A.  Mandel,  Professor  of 
Chemistry  and  Physics  and  Physiological  Chemistry  in  the 
New  York  University  and  Bellevue  Hospital  Medical  Col- 
lege.    8vo,  viii  -f-  703  pages',  cloth,  $4.00. 

A  Compendium  of  Chemistry,  Including  General,  inor- 
ganic, and  Organic  Chemistry. 

By  Dr.  Carl  Arnold,  Professor  of  Chemistry  in  the  Royal 
Veterinary  School  of  Hannover.  Authorized  translation 
from  the  eleventh  enlarged  and  revised  German  edition, 
by  John  A.  Mandel.  Small  8vo,  xii  +  627  pp.,  cloth, 
$3.50. 


A  TEXT-BOOK 


OP 


PHYSIOLOGICAL  CHEMISTRY. 


OLOF  HAMMARSTEN, 

Professor  of  Medical  am  Physiological  Chemistry  in  the 
University  of  Upaala. 


lUitbori^b  translation 

FROM  THE  A  UTHOR'S  ENLARGED  AND  REVISED 
FIFTH  GERMAN  EDITION 


JOHN   A.   MANDEL,   Sc.D., 

Professor  of  Chemistry  and  Physics,  and  of  Physiological  Chemistry,  (n  the 
New  York  University  and  Belle  cue  Hospital  Medical  College. 


FOURTH    EDITION. 
FIRST    THOUSAND. 


NEW    YORK: 

JOHN  WILEY  &  SONS. 

London:    CHAPMAN  &  HALL,   Limited. 
1904. 


QPSif- 

I4.it 

Ifcif- 

Copyright,  1900,  1904, 

BY 

JOHN  A.   MANDEL. 


ROBERT   DRTTMMONn.    PRINTER.   NEW  YORK. 


PREFACE  TO  THE  FIFTH   GERMAN  EDITION. 


The  numerous  publications  in  physiological  chemistry  which  have 
appeared  since  the  publication  of  the  last  edition  of  this  work,  and  the 
suggestion  of  new  methods  of  work,  have  necessitated  a  thorough  revision 
of  most  of  the  chapters.  As  stated  in  the  preface  to  the  second  edition, 
this  work  is  not  intended  as  a  complete  handbook,  but  only  as  a  rather 
short  textrbook;  it  was  my  desire  in  this  revision  to  prevent  a  too  great 
increase  in  the  size  of  the  book.  In  order  to  accomplish  this  I  have 
eliminated  in  part  certain  older,  superfluous,  or  at  present  untenable 
statements,  and  in  certain  instances  I  have  treated  the  chemical  methods 
of  work  less  fully  than  in  the  other  editions.  This  is  true  only  for 
those  methods  which  are  not  important  for  the  physician  and  student  or 
those  which  require  a  lengthy  detailed  description  and  which  can  be  found 
in  complete  works  on  chemical  analysis  or  in  the  original  publications. 
In  other  regards  the  plan  of  the  book  is  the  same  as  in  the  previous  editions. 

Dr.  S.  Schmidt-Nielsen  has  kindly  prepared  the  index. 

Olof  Hammarstkn. 
Upsala,  March  17,  1904. 

iii 


TRANSLATORS   PREFACE  TO  THE  FOURTH 
AMERICAN   EDITION. 


As  physiological  chemistry  has  made  such  rapid  advances  during  the 
last  five  years,  and  as  the  literature  of  the  subject  is  becoming  more  and 
more  specialized,  I  feel  confident  that  the  American  student  will  be  glad 
to  receive  the  present  edition,  and  I  hope  it  will  be  of  material  aid  in  the 
advancement  of  the  subject.  The  author's  addenda  have  been  incorpo- 
rated into  the  text. 

I  am  under  obligations  to  Dr.  Holmes  C.  Jackson  for  much  assistance 

in  proof  revision. 

John  A.  Mandel. 
New  York,  October,  1904. 


CONTENTS. 


CHAPTER  I. 

PA  OB 

Introduction 1 

CHAPTER  II. 
The  Protein  Substances 18 

CHAPTER  III. 
The  Carbohydrates 83 

CHAPTER  IV. 
The  Animal  Fats 108 

CHAPTER  V. 
The  Animal  Cells 116 

CHAPTER  VI. 
The  Blood 142 

CHAPTER  VII. 
Chyle,  Lymph,  Transudates,  and  Exudates 211 

CHAPTER  VIII. 
The  Liver 239 

CHAPTER  LX. 
Digestion 286 

CHAPTER  X. 

Testes  of  the  Connective  Substance 358 

vii 


viii  CONTENTS. 

CHAPTER  XI. 

PAGE 

The  Muscles • 376 

CHAPTER  XII. 
Brain  and  Nerves 406 

CHAPTER  XIII. 
Organs  of  Generation 419 

CHAPTER  XIV. 
The  Milk 437 

CHAPTER  XV. 
The  Urine 461 

CHAPTER  XVI. 
The  Skin  and  its  Secretions 588 

CHAPTER  XVII. 
Chemistry  of  Respiration 598 

CHAPTER  XVIII. 
Metabolism 616 

Index 673 


PHYSIOLOGICAL  CHEMISTRY. 


CHAPTER   I. 
INTRODUCTION. 

It  follows  from  the  law  of  the  conservation  of  matter  and  nf  pnp.rgv  that 
living  beings,  plants  and  animals,  can  produce  neither  new  matter  nor  new 
energy.  Thgy  are  only  called  upon  to  appropriate  and  assimilate  already 
existing  material  and  to  transform  it  into  ne.w  forms  of  energy. 

Out  of  a  few  relatively  simple  combinations,  especially  carbon  dioxide 
and_-ffia±er,  together  with  ammonium  compounds  or  nitrates,  and  a  few 
mineral  substances^  which  serve  as  its  food,  the  plant  builds  up  the 
extremely  complicated  constituents  of  its  organism,  proteids,  carbodydrates, 
fats,  resins,  organic  acids.,  etc.  The  chemical  work  which  is  performed  in 
theplant  must  therefore,  in  the  majority  of  cases,  consist  in  syntheses;  but 
besides  these,  processes  of  reduction  take  place  to  a  great  extent.  The 
radiant  energy  of  the  sunlight  induces  the  green  parts  of  the  plant  to  split 
off  oxygen  from  the  carbon  dioxide  and  water,  and  this  reduction  is  generally 
considered  as  the  starting-point  of  the  following  syntheses.  In  the  first 
jfl«r»ff  for^nalHphydp  is  prnrhmedr  (XL  4-  H20  =  CH2Q  +  Q?,  which  then  by 
condensation  is  transformed  into  sugar,  and  this  then  serves  in  the 
structure  of  other  bodies.  The_  energy  of  the  sun,  which  produces  this 
splitting,  is  not  lost;  it  is  only  transformed  and  is  stored  as  chemical 
energy  in  the  new  compounds  produced  in  the  synthesis. 

These  conditions  arenot  the  same  in  animals.  They  are  dependent 
either  directly,  as  the  herbivora,  or  indirectly,  as  the  carnivora,  upon  plant- 
life,  from  which  they  derive  the  three  chief  groups  of  organic  nutritive 
matter — proteins,  carbohydratps,  and  fats.  These  bodies,  of  which  the 
protein  substances  and  fats  form  the  chief  mass  of  the  animal  body,  undergo 
within  the  animal  organism  a  cleavage  and  oxidation^  and  yield  as  final 
product^  exactly  the  above-mentioned  chief  components  of  the  nutrition  of 
plants,  namely,  carbon  dioxide,  water,  and  ammonia  derivatives,  which  are 


2  INTRODUCTION. 

rich  in  oxygen  and  have  little  energy.  The  chemical  energy,  which  is 
partly  represented  by  the  free  oxygen  and  partly  stored  up  in  the  above- 
mentioned  more  complex  chemical  compounds,  is  transformed  into  other 
forms  of  energy,  namely,  heat  and  mechanica  work.  While  in  the  plant 
we  find  chiefly  reduction  processes  and  syntheses,  which  by  the  introduc- 
tion of  energy  from  without  produce  complex  compounds  having  a  greater 
content  of  energy,  we  find  in  the  animal  body  the  reverse  of  this,  namely 
cleavage  and  oxidation  processes,  which,  as  we  used  to  state,  convert 
chemical  tension  into  living  force. 

This  difference  between  animals  and  plants  must  not  be  overrated,  nor 
must  we  consider  that  there  exists  a  sharp  boundary-line  between  the  two. 
This  is  not  the  case.  There  are  not  only  lower  plants,  free  from  chloro- 
phyll, which  in  regard  to  chemical  processes  represent  intermediate  steps 
between  higher  plants  and  animals,  but  the  difference  existing  between  the 
higher  plants  and  animals  is  more  of  a  quantitative  than  a  qualitative  kind. 
Plants  require  oxygen  as  peremptorily  as  do  animals  Like  the  animal,  the 
plant  also,  in  the  dark  and  by  means  of  those  parts  which  are  free  from 
chlorophyll,  takes  up  oxygen  and  eliminates  carbon  dioxide,  while  in  the 
light  the  oxidation  processes  going  on  in  the  green  parts  are  overshadowed 
or  hidden  beneath  the  more  intense  reduction  processes.  Like  the  animal 
we  also  find  a  heat  production  in  fermentation  produced  by  plant  organisms ; 
and  even  in  a  few  of  the  higher  plants — as  the  aroideoB  when  bearing 
fruit — a  considerable  development  of  heat  has  been  observed.  The  reverse 
is  found  in  the  animal  organism,  for,  besides  oxidation  and  splitting,  reduc- 
tion processes  and  syntheses  also  take  place.  The  contrast  which  seemingly 
exists  between  animals  and  plants  consists  merely  in  that  in  the  animal 
organism  the  processes  of  oxidation  and  splitting  are  prevalent,  while  in  the 
plant  chiefly  those  of  reduction  and  synthesis  have  thus  far  been  observed. 

Wohler1  in  1824  furnished  the  first  example  of  synthetical 
processes  within  the  animal  organism.  He  showed  that  when  benzoic  acid 
is  introduced  into  the  stomach  it  reappears  as  hippuric  acid  in  the  urine, 
after  it  combines  with  glycocoll  (aminoacetic  acid).  Since  the  discovery 
of  this  synthesis,  which  may  be  expressed  by  the  following  equation: 

C6H„.COOH  +  NH2.CH2.COOH  =  NH(C6H5.CO)  .CH2.COOH + H20, 

Benzoic  acid  Glycocoll  Hippuric  acid 

and  which  is  ordinarily  considered  as  a  type  of  an  entire  series  of  syntheses 
occurring  in  the  body  where  water  is  eliminated,  the  number  of  known 
syntheses  in  the  animal  kingdom  has  increased  considerably.  Many  of 
these  syntheses  have  also  been  artificially  produced  outside  of  the  organism, 
and  numerous  examples  of  animal  syntheses  of  which  the  course  is  abso- 
lutely clear  will  be  found  in  the  following  pages.     Besides  these  well-studied 


1  Berzelius,  Lehrb.  d.  Chemie,  ubersetzt  von  Wohler,  4,  S.  356,  Abt.  1 ,  Dresden,  1831. 


ANIMAL  OXIDATIONS.  3 

syntheses,  there  occur  in  the  animal  body  also  similar  processes  unquestion- 
ably of  the  greatest  importance  to  animal  life,  but  of  which  we  know 
nothing  with  positiveness.  We  enumerate  as  examples  of  this  kind  of 
synthesis  the  re-formation  of  the  red-blood  pigment  (the  haemoglobin),  the 
formation  of  the  different  proteids  from  simpler  substances  and  the  produc- 
tion of  fat  from  carbohydrates.  This  last-mentioned  process,  the  formation 
of  fat  from  carbohydrates,  is  also  an  example  of  reduction  processes  which 
occur  to  a  considerable  extent  in  the  animal  body. 

Formerly  the  view  was  generally  accepted  that  animal  oxidation  took 
place  in  the  fluids,  while  to-day  we  are  of  the  opinion,  derived  from  the 
investigations  of  Pfluger  and  his  pupils,1  that  it  is  connected  with  the 
form-elements  and  the  tissues.  The  question  how  this  oxidation  in  the 
form-elements  proceeds  and  how  it  is  induced  cannot  be  answered  with 
certainty. 

When  a  body  is  oxidized  by  neutral  oxygen  at  ordinary  temperature  or 
at  the  temperature  of  the  body,  the  body  is  called  easily  oxidized  or  auto- 
oxidized  and  the  process  is  considered  as  a  direct  oxidation  or  autooxidation. 
As  the  oxygen  of  the  inspired  air,  and  that  of  the  blood,  is  neutral,  molecular 
oxygen,  the  old  assumption  that  ozone  occurs  in  the  organism  has  now  been 
discarded  for  several  reasons.  On  the  other  hand  the  chief  groups  of 
organic  nutritives,  carbohydrates,  fat,  and  proteids,  the  last  two  forming 
the  chief  mass  of  the  animal  body,  are  not  autooxidizable  substances.  They 
are  on  the  contrary  bradoxidizable  (Traube)  or  dysoxidizable  bodies. 
They  are  nearly  indifferent  to  neutral  oxygen,  and  it  is  therefore  a  question 
how  an  oxidation  of  these  and  other  dysoxidizable  bodies  is  possible  in  the 
animal  body. 

In  explanation  it  is  very  generally  admitted  that  the  oxygen  is  made 
activeand  this  causes  a  secondary  oxidation.  It  is  generally  conceded  that 
in  autooxidation  a  cleavage  of  neutral  oxygen  takes  place.  The  autooxidiz- 
able substance  splits  the  oxygen  molecule  and  combines  with  one  of  the 
oxygen  atoms,  while  the  other  free  atom  as  active  oxygen  may  oxidize  the" 
dysoxidiyfl.blg  siihstanops  simultaneously  present.  Such  a  subordinate  oxi- 
dation is  called  an  indirect  or  secondary  oxidation.  The  explanation  of 
animal  oxidations  has  been  attempted  in  different  ways  by  the  supposition 
that  the  oxygen  is  made  active  and  thus  produces  secondary  oxidation. 

The  cause  of  the  animal  oxidation  is  considered,  by  Pfluger  and 
several  other  investigators,  to  be  dependent  upon  the  special  constitution  of 
the  ^protoplasmic  proteids  or  the  living  protr>plfl^Tr)jn  substance.  This 
investigator  calis  the  proteids  outside  of  the  organism,  or  those  which 
occur  in  the  animal  fluids,  "non-living  proteids,"  or  at  least  somewhat 
different    from    those    occurring    in    living  protoplasm — "living  proteids" 

1  Pfluger.  Pfluger's  Archiv,  6  and  10;  Finkler,  Aid.,  10  and  14;  Oertman,  ibid., 
14  and  15;  Hoppe-Seyler,  ibid.,  7. 


4  INTRODUCTION. 

(Pfluger),  "active  proteids"  (Loew),  or  "biogens"  (Verworn).  The 
living  protoplasmic  molecule  differs  from  the  ordinary  non-living  proteid  by 
being  more  unstable  and  therefore  having  a  greater  inclination  towards 
intramolecular  changes  of  the  atoms.  The  reason  for  these  greater  intra- 
molecular movements  Pfluger  ascribes  to  the  presence  of  cyanogen,  and 
Latham  attributes  it  to  the  presence  of  a  chain  of  cyanalcohols  in  the 
proteid  molecule.  Verworn,1  on  the  contrary,  claims  an  intramolecular 
introduction  of  oxygen  into  a  large  hypothetical  protoplasmic  molecule, 
the  "biogen  molecule,"  which,  as  oxygen  receptor  or  translator  of  a  nitrogen 
or  iron  compound,  contains  as  oxidation  material  a  side  chain  constructed 
like  the  carbohydrates  with  aldehydic  character. 

According  to  Loew,2  who  bases  his  claim  upon  special  investigations 
and  numerous  toxicological  observations,  the  unstability  of  the  active 
proteid  molecule  is  due  to  the  simultaneous  presence  of  aldehyde-  and 
unstable  amino-groups.  These  occur  separated  from  each  other  in  the 
active  proteids,  and  when  they  combine  the  protoplasm  dies,  the  molecule 
being  changed  into  a  stable  condition,  i.e.,  into  dead  proteid.  It  is  also  a  fact 
that  all  substances  which  react  with  aldehyde-  and  unstable  amino-groups 
are  poisonous  to  the  living  cells. 

Loew  has  also,  in  conjunction  with  Bokorny,  shown  that  in  many 
plants  a  very  unstable  reserve-protein  substance  occurs,  which  to  a  certain 
extent  occupies  an  intermediate  position  between  proteid  and  organized 
living  substance. 

The  explanation  as  to  the  oxidation  process  is  entirely  different  accord- 
ing to  our  conception  of  the  structure  of  the  unstable  protoplasmic  mole- 
cule. If  the  living  protoplasmic  proteid  is  not,  like  proteid  in  the  ordinary 
sense,  indifferent  to  neutral  oxygen,  we  can  admit  of  a  cleavage  of  the 
oxygen  molecule  by  this  change.  The  proteid  would  be  oxidized  itself, 
while  on  the  other  hand  a  secondary  oxidation  of  other  difficultly  oxidiz- 
able  substances  could  be  brought  about  by  the  oxygen  atoms  set  free. 

Another  very  widely  diffused  view  exists  in  regard  to  the  origin  of  the 
activity  of  the  oxygen,  namely,  that  by  the  decomposition  processes  in  the 
tissues,  reducing  substances  are  formed  which  split  the  neutral  oxygen 
molecule,  uniting  with  one  oxygen  atom  and  setting  the  other  free. 

The  formation  of  reducing  substances  during  fermentation  and  putre- 
faction is  generally  known.  The  butyric  fermentation  of  dextrose  in  which 
hydrogen  is  set  free  — C6H120(i=C4H802+2C02+2H,  — is  an  example  of 
this  kind.  Another  example  is  the  appearance  of  nitrates  in  consequence 
of  an  oxidation  of  nitrogen  in  cases  of  putrefaction,  which  process  is  ordi- 

1  Pfli'iger,  Pfluger's  Archiv,  10;  Latham,  Brit.  Med.  Journal,  1886;  Verworn, 
"Die  Biogenhypothese,"  Jena,  1903. 

2  Loew  and  Bokorny,  Pfluger's  Archiv,  25;  O.  Loew,  ibid.,  30g  and  specially 
O.  Loew,  "The  Energy  of  Living  Protoplasm,"  London,  1896. 


ANIMAL  OXIDATIONS.  5 

narily  explained  by  the  statement  that  reducing,  easily  oxidizable  bodies 
are  formed  which  split  oxygen  molecules,  liberating  oxygen  atoms  which 
afterward  oxidize  the  nitrogen.  It  is  assumed  also  that  the  cells  of  the 
animal  tissues  and  organs  have  the  property  like  these  lower  organ  i.»n  is, 
which  produce  fermentation  and  putrefaction,  of  causing  splitting  processes 
in  which  easily  oxidizable  substances,  perhaps  also  nascent  hydrogen 
(Hoppe-Seyler  '),  are  produced. 

In  accordance  with  what  has  been  stated  above  on  the  oxidation-  of 
theanimal  body,  primarily  a.  oWvage  of  the  organic  constituents  of  the 
body  takes  place  with  the  formation  of  readily  oxidizable  substances. 
The  oxidation  of  these  latter  produces  an  activation  of  the  oxygen  and 
hence  may  also  cause  a  secondary  oxidation  of  dysoxidizable  substances. 
The  products  formed  by  these  splittings  and  oxidations  may  perhaps  in 
part  be  burned  within  the  body  without  undergoing  further  cleavage,  but 
more  probably  they  must  first  undergo  a  further  cleavage  and  then  succumb 
to  consecutive  oxidation,  until  after  repeated  cleavage  and  oxidation  the 
final  products  of  metabolism  are  formed 

An  activation  of  the  oxygen  may  be  produced  according  to  0.  Nasse2  by  a 
hydroxylization  of  the  constituents  of  the  protoplasm  with  the  splitting  off  of 
molecules  of  water.  If  benzaldehyde  is  shaken  with  water  and  air,  an  oxidation 
of  the  benzaldehj'de  into  benzoic  acid  takes  place,  while  oxidizable  substances 
present  at  the  same  time  may  also  be  oxidized.  The  simultaneous  presence  of  po- 
tassium iodide  and  starch  or  tincture  of  guaiacum  causes  a  blue  coloration  because 
the  hydroxy!  (OH)  takes  the  place  of  the  hydrogen  in  the  aldehyde-group,  and 
these  two  hydrogen  atoms,  one  derived  from  the  aldehyde  and  the  other  from 
the  water,  have  a  splitting  action  on  the  molecular  oxygen.  Nasse  and  Rosing' 
have  also  found  that  certain  varieties  of  proteid  have  the  property  of  being 
hydroxylized  in  the  presence  of  water.  According  to  Nasse  a  whoie  series  of 
oxidations  in  the  animal  body  may  be  accounted  for  by  the  oxygen  atoms  set 
free  in  the  hydroxylization  similar  to  that  of  benzaldehyde.  In  opposition  to 
this  view  we  must  remark  that  the  oxidation  of  benzaldehyde  to  benzoic  acid 
may  also  take  place  in  other  ways,  thus  by  the  intermediary  formation  of  a  per- 
oxide (see  Baeyer  and  Villiger;   Engler  and  Weissberg4). 

By  quantitative  methods  van  't  Hoff  and  his  pupils  5  have  shown 
that  molecular  oxygen  can  be  divided  in  two  parts  by  certain  autooxida- 
tion  processes.  One  of  these  unites  with  the  autooxidizer  and  the  other 
with  a  body  simultaneously  present  but  not  directly  oxidizable,  which,  ac- 
cording to  the  suggestion  of  Engler,6  is  called  the  acceptor,     van 't  Hoff 

1  Pfluger's  Archiv,  12. 

20.  Nasse,  Rostocker  Zeitung,  No.  534,  1891,  and  No.  363,  1895. 

3  E.  Rosing,  Untersuchungen  iiber  die  Oxydation  von  Eiweiss  in  Gegenwart  von 
Schwefel.     Inaug.  Dissert.     Rostock,  1891. 

4  Baeyer  and  Villiger,  Ber.  d.  d.  chem.  Gesellsch.,  33;  Engler  and  Weissberg, 
ibid.,  33. 

5  van't  Hoff,  Zeitschr.  f.  physikal.  Chem.,  16;  Jorissen,  Ber.  d.  d.  chem.  Gesellsch., 
30,  and  Zeitschr.  f.  physikal.  Chem.,  22;   Ewan.,  ibid.,  16. 

*Ber.  d.  d.  chem.  Gesellsch.,  33. 


6  INTRODUCTION. 

claims  that  the  oxygen  molecule  dissociates  at  ordinary  temperatures  into 
minimum  quantities  of  positively  and  negatively  charged  oxygen  atoms, 
the  ions  of  similar  charge  uniting  with  the  autooxidizable  substance, 
while  the  remaining  ions  oxidize  the  acceptor.  Such  a  division  of  the 
oxygen  into  two  halves  has  also  been  shown  by  other  investigators  such 
as  Manchot,  Engler,  and  his  collaborators.1  These  investigators  never- 
theless consider  that  autooxidation  takes  place  in  another  way,  namely,  by 
the  formation  first  of  peroxides   by  the  taking  up  of  oxygen  molecules. 

Traube  2  has  also  expressed  a  similar  view.  According  to  him,  in 
autooxidation  we  have  to  deal  in  the  first  place,  not  with  a  cleavage  of  the 
oxygen,  but  with  a  splitting  of  water  in  which  the  hydroxyl  groups  of  the 
water  combine  with  the  oxidizable  substance,  while  the  hydrogen  atoms 
set  free  on  the  decomposition  of  the  water  unites  with  the  neutral  oxygen, 
forming  hydrogen  peroxide,  which  may  naturally  also  have  an  oxidizing 

action. 

A+H20  +  02=A(OH)2+H202. 

According  to  the  view  of  Engler  and  his  collaborators,  which  corre- 
sponds in  great  measure  with  those  of  Bach  and  of  Manchot,3  at  least  in 
the  simplest  cases  ("direct  autooxidation"  according  to  Engler)  the 
oxygen  molecules  unite  with  the  activating  body  (A),  forming  a  peroxide- 
like substance  which  can  give  up  one  of  the  two  oxygen  atoms  to  an  accep- 
tor (B): 

A+02=A02    and    A02+B=AO+BO. 

If  this  is  so,  still  we  do  not  know  to  what  extent  such  peroxides  are 
formed  in  the  oxidation  in  the  living  cell.  The  possibility  of  a  production 
of  peroxides,  or  also  hydrogen  peroxide,  in  animal  oxidation  is  still  gener- 
ally admitted,  and  Chodat  and  Bach  4  have  indeed  been  able  to  show  a 
peroxide  formation  in  plants.  Still,  if  hydrogen  peroxide  were  formed 
in  such  oxidations  it  would  have  no  further  physiological  importance, 
according  to  Loew,  because  the  animal  and  plant  cells  contain  special 
enzymes,  called  by  him  catalases,  which  quickly  decompose  the  hydrogen 
peroxide  with  the  production  of  molecular  oxygen.  According  to  Loew  5 
the  physiological  importance  of  the  catalases  is  to  protect  the  cell  from 
hydrogen  peroxide,  which  acts  as  a  protoplasmic  poison. 

'Manchot,  Uber  freiwillige  Oxydation,  Leipzig,  1900;  Engler  and  Weissberg, 
Ber.  d.  d.  chera.  Gesellsch.,  33;    Engler  and  Frankenstein,  ibid.,  34. 

2  Ber.  d.  d.  chem.  Gesellsch.,  15,  18,  19,  22,  and  26. 

3  Engler  and  Wild,  -ibid.,  30;  Bach,  Le  Moniteur  scientifique,  1897,  and  Compt. 
rend.,  124;   Manchot,  1.  c. 

4  Ber.  d.  d.  chem.  Gesellsch.,  35  u.  36. 

5  Loew,  U.  S.  Dept.  of  Agriculture,  Rep.  68,  1901,  and  Ber.  d.  d.  chem.  Gesellsch., 
35;  in  regard  to  the  opposed  views  see  Chodat  and  Bach,  1.  c,  and  Kastle  and  Loeven- 
hart,  Amer.  Chem.  Journ.,  29. 


ANIMAL  OXIDATIONS.  7 

Loew,1  who  has  opposed  the  view  as  to  the  oxygen  becoming  active 
with  the  setting  free  of  oxygen  atoms,  has  sought  for  the  reason  of  the 
oxidations  in  the  unstable  properties  of  the  protoplasmic  proteids.  The 
active  movement  of  the  atoms  within  the  active  proteid  molecule  is  trans- 
mitted to  the  oxygen  and  to  the  oxidizable  substance,  and  when  the  disso- 
lution of  the  molecule  has  proceeded  to  a  certain  point  the  oxidation  occurs 
by  the  chemical  affinity.  The  reason  for  this  unstable  condition  of  living 
proteid  molecules  has  already  been  given  above. 

Schmiedeberg,2  who  also  denies  the  supposition  that  the  oxygen 
becomes  active,  is  of  the  view  that  the  tissues  by  the  mediation  of  the  oxida- 
tions do  not  increase  the  oxidizing  activity  of  the  oxygen,  but  more  probably 
act  on  the  oxidizing  substances,  making  them  more  accessible  to  oxidation. 

All  the  views  presented  thus  far  assume  a  continuous  oxidation  of  the 
primary  active  substance.  The  view  has  also  been  suggested  that  animal 
oxidation  may  be  brought  about  by  oxygen-carriers,  i.e.,  by  bodies  which, 
according  to  the  older  views,  without  being  oxidized  themselves,  act  in  an 
analogous  manner  to  the  nitric  oxide  in  the  manufacture  of  sulphuric  acid 
by  alternately  taking  up  and  introducing  oxygen  in  the  oxidation  of  dys- 
oxidizable  bodies.  Traube  has  for  a  long  time  explained  the  oxidations 
of  the  animal  body  in  this  way,  and  he  calls  these  questionable  oxygen- 
carriers  oxidation  ferments.3 

It  has  also  been  positively  proved  by  the  researches  of  Jaquet,  Sal- 
kowski,  Spitzer,  Rohmann,  Abelous  and  Biarnes,  Bertrand,  Bour- 
quelot,  De  Rey-Pailhade,  M edvedew,  Pohl,  Jacoby,  Chodat  and  Bach/ 
and  others  that  in  the  blood  and  different  tissues  of  the  animal  body,  as  also 
in  plant-cells,  substances  occur  which  have  the  property  of  causing  certain 
oxidations  and  are  therefore  called  oxidation  ferments  or  oxidases.  Little 
is  known  in  regard  to  the  nature  or  the  manner  of  action  of  these  bodies. 
Certain  of  these  oxidases  are  nucleoproteids  (Spitzer),  and  others,  like  the 
catalases  (Loew),  are  proteoses,  while  others,  on  the  contrary,  like  the 
liver  aldehydase  (Jacoby)  and  laccase  (Bertrand),  are  not  of  proteid 
nature.  A  large  number  of  these  oxidases,  so-called  direct  or  primary 
oxidases,  turn  tincture  of  guaiacum  blue  directly.  Others,  on  the  con- 
trary, the  indirect  oxidases  or  peroxidases,  decompose  hydrogen  peroxide 

1  O.  Loew,  The  Energy  of  Living  Protoplasm,  London,  1896. 
'  Arch.  f.  exp.  Path.  u.  Pharm.,  14. 

3  M.  Traube,  Theorie  der  Fermentwirkungen.     Berlin,  1858.  . 

4  Jaquet,  Arch.  f.  exp.  Path.  u.  Pharm.,  29;  Salkowski,  Centralbl.  f.  d.  med.  Wis- 
sensch.,  1892  and  1S94,  and  Yirchow's  Arch.,  147;  Spitzer,  Pfliiger's  Archiv,  60  and 
67;  Spitzer  and  Rohmann,  Ber.  d.  deutsch.  chem.  Gesellsch.,  2S;  Abelous  et  Biarnes, 
Arch,  de  physiol.  (5),  7,  8,  and  9,  and  Compt.  rend.  soc.  biol.,  46;  Bertrand,  Arch,  de 
physiol.  (5),  8,  9,  and  Compt.  rend.,  122,  123,  124;  Bourquelot,  Compt.  rend.  soc. 
biol.,  48,  and  Compt.  rend.,  123;  De  Rey-Pailhade,  1.  c  ;  Medvedew,  Pfliiger's  Arch., 
65;  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  38;  Jacoby,  Ergebnisse  der  Physiologie, 
Jahrg.  I,  Abt.  I,  which  contains  the  literature  of  the  subjectj  Chodat  and  Bach,  1.  c. 


8  INTRODUCTION. 

and  only  turn  tincture  of  guaiacum  blue  in  the  presence  of  peroxides, 
while  others,  like  the  catalases,  which  are  called  oxidases  by  Loew,  decom- 
pose hydrogen  peroxide  with  activity,  but  do  not  turn  tincture  of  guaiacum 
blue  either  directly  or  indirectly  in  the  presence  of  peroxides. 

The  substances  upon  which  these  oxidases  act  may  at  the  same  time 
be  of  the  greatest  difference.  Thus  the  oxidases  studied  by  Rohmann  and 
Spitzer  may  by  synthetical  oxidation  produce  indophenol  from  a-naphthol 
and  p-phenylendiamine  in  the  presence  of  alkali.  The  salicylase  or  alde- 
hydase  detected  in  the  liver  and  many  other  organs  oxidizes  many  alde- 
hydes to  their  corresponding  acid,  but  does  not  give  the  indophenol  reaction. 
The  laccase  isolated  by  Bertrand  from  the  juice  of  the  lac-tree  has  an 
oxidizing  action  upon  polyhydric  p-phenols,  such  as  hydroquinone,  but 
not  upon  tyrosin.  The  bodies  called  tyrosinases  first  found  by  Bertrand 
in  certain  fungi  and  later  also  found  by  Biedermann,  v.  Furth  and 
Schneider  in  the  animal  kingdom  have,  on  the  contrary,  an  action  upon 
tyrosin  converting  it  into  homogentisic  acid  (Gonnermann  *)  or  other 
colored  compounds. 

At  present  we  know  only  very  little  with  certainty  in  regard  to  the 
mode  of  action  of  these  oxidases.  It  is  generally  admitted  that  we  are 
here  dealing  with  a  catalysis  produced  by  intermediate  reactions.  As  in 
certain  oxidations  manganous  and  ferrous  salts  act  as  catalysators,  so  an 
important  role  as  oxygen-carriers  has  been  ascribed  to  these  metals,  espe- 
cially in  laccase  which  contains  manganese,  and  in  the  oxidases,  containing 
iron  (Spitzer 's  nucleoproteid).  Manchot  2  in  his  work  on  the  autooxida- 
tion  of  ferrous  sulphate  has  recently  called  attention  to  the  apparently 
great  importance  of  iron  for  the  physiological  oxidations. 

According  to  the  observations  of  Chodat  and  Bach  3  upon  plants,  the  oxidases 
are  a  mixture  of  oxygenases  and  peroxidases.  The  oxygenases  are  of  a  proteid 
nature  and  contain  manganese  or  iron  and  are  converted  into  peroxides  by  the 
taking  up  of  oxygen.  These  peroxides  themselves  only  have  a  slight  oxidizing 
power,  but  are  made  active  by  the  peroxidases  in  a  manner  similar  to  the  activ- 
ation of  hydrogen  peroxide  by  platinum-black  and  ferrous  sulphate.  The  per- 
oxidases, which  do  not  have  the  slightest  oxidizing  power  in  the  absence  of  per- 
oxides, are  not  proteids.  The  catalases,  which  decompose  hydrogen  peroxide  with 
the  development  of  molecular  oxygen,  also  belong  to  the  enzymes  taking  part 
in  the  oxidation  processes.  In  oxidation,  according  to  the  hypothesis  of  Chodat 
and  Bach,  the  molecular  oxygen  is  first  converted  by  the  oxygenase  into  per- 
oxide. The  peroxide  is  activated  by  the  peroxidase  and  then  has  powerful 
oxidizing  power.  The  catalase  decomposes  the  hydrogen  peroxide,  which  has  a 
destructive  action  and  which  is  abundantly  formed  under  certain  circumstances. 

Like  the  other  enzymes  the  oxidases  also  have  a  pronounced  specific 
action,  as,  for  example,  a  certain  oxidase,  like  laccase,  only  oxidizes  certain 

1  Biedermann,  Pfluger's  Arch.,  72;  v.  Furth  and  Schneider,  Hofmeister's  Beitr. 
z.  chem.  Phys.  u.  Path.,  1;  Gdnnermann,  Pfluger's  Arch.,  82. 

2  Zeitschr.  f.  anorg.  Chem.,  27. 

3  See  Bioch.  Centralbl..  1.  417  and  457. 


ANIMAL  OXIDATIONS.  9 

substances,  but  not  others.  This  behavior,  which  is  difficult  of  explanation 
according  to  the  hypothesis  of  Chodat  and  Bach,  shows,  according  to 
Medwedew,1  that  the  substances  which  are  active  in  oxidation  do  not 
act  upon  the  oxygen,  but  rather  upon  the  substance  to  be  oxidized.  At 
present  it  is  difficult  to  say  how  far  special  oxidation  enzymes  are  active 
in  the  oxidations  in  the  living  animal  body. 

Thc  many  different  views  in  regard  to  the  oxidation  processes  show- 
US  strikingly  how  little  is  positively  known  about  these  processes.  The 
occurrence  of  numerous  intermediary  decomposition  products  in  the  animal 
body  teaches  us  that  the  oxidation  of  the  constituents  of  the  body  is  not 
instantaneous  and  sudden,  but  takes  place  step  by  step,  and  hand  in  hand 
with  cleavages.  Most  investigators  are  agreed  that  these  decompositions 
are  similar  to  certain  oxidations  studied  by  Drechsel  2  outside  the  animal 
body,  where  oxidations  and  reductions  alternate  in  quick  succession.  The 
views  are  divided  in  regard  to  the  manner  and  origin  of  this  cooperative 
action.3 

The  oxidations  in  the  animal  body  have  long  been  designated  as  a  com- 
bustion, and  such  a  conception  is  easily  reconcilable  with  the  above-mentioned 
views.  In  combustion  in  the  ordinary  sense,  as,  for  example,  the  burning 
of  wood  or  oil,  we  must  not  forget  that  the  substances  themselves  do  not 
combine  with  oxygen.  It  is  only  after  the  action  of  heat  has  decomposed 
these  bodies  to  a  certain  degree  that  the  oxidation  of  the  products  of  such" 
decomposition  takes  place  and  is  accompanied  by  the  phenomenon  of  light7~ 

The  essential  source  of  heat  and  mechanical  work  developed  in  the 
organism  is  to  be  found  in  the  oxidations.  Chemical  energy  is  transformed 
into  the  above-mentioned  forms  of  energy  in  cleavage  processes,  where 
complicated  chemical  compounds  are  reduced  to  simpler  ones,  and  there- 
fore the  atoms  change  from  a  unstable  to  a  stabler  equilibrium,  ancT 
stronger  chemical  affinities  are  satisfied.  The  best-known  example  of  such 
a  splitting  process  is  the  ordinary  alcoholic  fermentation  of  dextrose, 
C6H,206  =  2C02  -I-  2C2H60,  a  process  which,  according  to  the  very  interesting 
investigations  of  Stoklasa  and  his  collaborators,4  occurs  also  in  animal 
life  in  anaerobic  respiration.  The  animal  body  may  also  have  a  source  of 
energy  in  the  cleavage  processes  which  are  not  dependent  on  the  presence 
of  free  oxygen.  The  processes  taking  place  in  the  living  muscle  are  an 
example  of  this  kind.  A  removed  muscle,  which  gives  no  oxygen  when  in 
a  vacuum,  may,  as  Hermann  5  has  shown,  work,  at  least  for  a  time,  in  an 

'Pfliiger's  Arch.,  81. 

2  Journ.  f.  prakt.  Chem.  (N.  F.),  22,  29,  38,  and  Festschrift  fur  C.  Ludwig,  1887. 

3  See  M.  Nencki,  Arch,  des  sciences  biol.  de  St.  Petersbourg,  1,  483. 

4  Hofmeister's  Beitr.,  3,  and  Centralbl.  f.  Physiol.,  16,  652  and  712.  See  also 
Stoklasa.  Osterreich.  Chem.  Zeitung,  1903,  and  Centralbl.  f.  Physiol.,  17;  Stoklasa  and 
Czcrny,  Ber.  d.  d.  chem.  Gesellsch.,  36;  Blumenthal,  Deutsch.  Med.  Wochenschr., 
1903;    Feinschmidt,  Hofmeister's  Beitriige,  4. 

6  Untersuch.  iiber  den  Stoffwechsel  der  Menschen,  Berlin,  1867. 


10  INTRODUCTION. 

atmosphere  devoid  of  oxygen,  and  give  off  carbon  dioxide  at  the  same 
time. 

Cleavage  processes  which  are  accompanied  by  a  decomposition  of  water 
and  then  a  taking  up  of  its  constituents  are  called  hydrolytic  cleavages.  These 
cleavages,  which  play  an  important  role  within  the  animal  body,  and  which 
are  most  frequently  met  with  in  the  processes  of  digestion,  are,  for  example, 
the  transformation  of  starch  into  sugar  and  the  splitting  of  neutral  fats 
into  the  corresponding  fatty  acid  and  glycerine; 

aH5(C18H3503)3  +  3H20  =  C3H5(OH)3  +  3(C18H3602) . 

Tristearin  Glycerine  Stearic  acid 

As  a  rule  the  hydrolytic  cleavage  processes  as  they  occur  in  the  animal 
body  may  be  performed  outside  of  it  by  means  of  higher  temperatures  with 
or  without  the  simultaneous  action  of  acids  or  alkalies.  Considering  the 
two  above-mentioned  examples,  we  know  that  starch  is  converted  into 
sugar  when  it  is  boiled  with  dilute  acids,  and  also  that  the  fats  are  split 
into  fatty  acids  and  glycerine  on  heating  them  with  caustic  alkalies  or  by 
the  action  of  superheated  steam.  The  heat  or  the  chemical  reagents  which 
are  used  for  the  performance  of  these  reactions  would  cause  immediate 
death  if  applied  to  the  living  body.  Consequently  the  animal  organism 
must  have  other  means  at  its  disposal  which  act  similarly,  but  in  such  a 
manner  that  they  may  work  without  endangering  the  life  or  normal  con- 
stitution of  the  tissues.  Such  means  have  been  recognized  in  the  so-called 
unorganized  ferments  or  enzymes. 

Alcoholic  fermentation,  as  well  as  other  processes  of  fermentation  and 
putrefaction,  is  dependent  upon  the  presence  of  living  organisms,  ferment 
fungi,  and  splitting  fungi  of  different  kinds.  The  ordinary  view,  according 
tn_thp  rp^e,fl,rp.lips  <~>f  Pasjf,ttr,  is  that  these  processes  are  to  be  considered  as 
phases  of  life  of  these  organisms.  The  name  organized  ferments  or  ferments 
has  been  p;iven  to  sno.h  minro-organisms  of  which  ordinary  yeast  is  an 
example.  However,  the  same  name  has  also  been  given  to  certain  bodies  or 
mixtures  of  bodies  of  unknown  organic  origin  which  are  products  of  the 
chemical  work  within  the  cell,  and  which  after  they  are  removed  from  the 
cell  still  have  their  characteristic  action.  Such  bodies,  for  example  malt 
diastase,  rennin,  and  the  digestive  ferments,  are  capable  in  the  very  small- 
est  quantity  of  causing  a  decomposition  or  cleavage  in  very  considerable 
quantities  of  other  substances  without  entering  into  permanent  chemical 
combination  with  the  decomposed  body  or  with  any  of  the  cleavage  or 
decomposition  products.  Thase  formless  or  unorganized  ferments  are 
generally  called  enzymes,  according  to  Kuhne. 

A  ferment  in  a  more  restricted  sense  is  therefore  a  living  being,  while 
an  enzyme  is  a  product  of  chemical  processes  in  the  cell,  a  product  which 
has  an  individuality  even  without  the  cell,  and  which  may  be  active  when 
separated  from  the  celh  The  splitting  of  invert-sugar  into  carbon  dioxide 
and  alcohol  by  fermentation  is  a  fermentative  process  closely  connected 


FERMENTS  AND  ENZYMES.  11 

with  the  life  of  the  yeast,  The  inversion  of  cane-sugar  is,  on  the  contrary, 
an  enzymotic  process  caused  by  one  of  the  bodies  or  mixture  of  bodies 
formed  by  the  living  ferment,  which  can  be  severed  from  this  ferment,  and 
still  remain  active  even  after  the  death  of  the  latter.  Consequently  fer- 
ments and  enzymes  are  capable  of  manifesting  a  different  behavior  towards 
certain  chemical  reagents.  Thus  there  exist  a  number  of  substances, 
among  which  we  may  mention [arsenious  acid,  phenol,  toluene,  salicylic 
acid,  boracic  acid,  sodium  fluoride,  chloroform,  ether]]  and  others,  which 
in  certain  concentration  kill  ferments,  but  which  do  not  noticeably  impair 
the  action  of  the  enzymes. 

The  above  view  as  to  the  difference  between  ferments  and  enzymes  has 
lately  been  essentially  shaken  by  the  researches  of  E.  Buchner  !  and  his 
pupils.  He  has  been  able  to  obtain  from  beer-yeast,  by  grinding  and 
strong  pressure,  a  cell  fluid  rich  in  proteid  which  when  introduced  into 
a  solution  of  a  fermentable  sugar  caused  a  violent  fermentation.  The 
objections  raised  from  several  sides  that  the  fluid  expressed  still  contained 
dissolved  living  cell  substance  has  been  so  successfully  answered  by  Btjch- 
ner  and  his  collaborators,  that  there  is  at  present  no  question  but  that 
alcoholic  fermentation  is  caused  by  a  special  enzyme  called  zymase  which 
is  formed  in  the  yeas^-cell. 

As  from  the  yeast-cell  so  also  from  other  lower  organisms,  indeed  from 
the  lactic-acid  bacilli  and  beer-vinegar  bacteria.,  we  have  recently  been  able 
to  isolate  enzymes  (E.  Buchxer  and  Meisexheimer,  Herzog  '-')  which 
produce  the  specific  fermentative  action  of  the  mother  organism.  We 
have  therefore  now  no  foundation  for  a  sharp  differentiation  between  the 
organized  ferments  and  the  enzymes. 

Many  enzymes  are  secreted  by  the  cells  and  are  therefore  called  secre- 
tion enzymes.  These  do  not  seem  to  be  secreted  as  such,  but  more  likely 
occur  as  precursors  of  the  enzymes,  the  zymogens,  in  the  cells.  These 
zymogens  are  then  transformed  by  special  influence  into  the  enzymes 

Besides  these  extracellular  enzymes  we  also  find  others  which  are  active 
within  the  cells,  the  intracellular  enzymes.  To  this  group  belongs  a  large 
number  of  enzymes,  among  which  are  those  proteolytic  enzymes  first 
observed  by  Salkowski  and  his  pupils,  which  produce  post-mortem  self- 
digestion  of  different  organs^  which  he  called  autodigestion.  Jacop.y  has 
recently  further  studied  this  autodigestion  and  has  called  it  autolysis.     We 

1  E.  Buchner,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30  and  31;  E.  Buchner  and  Rapp, 
ibid.,  31,  32,  34;  H.  Buchner,  Sitzungsber.  d.  Gesellsch.  f.  Morphol.  u.  Physiol,  in 
Miinchen,  13,  1897,  part  1,  which  also  contains  the  discussion  on  this  topic.  See  also 
Stavenhagen,  Ber.  d.  deutsch.  Chem.  Gesellsch.,  30;  Albert  and  Buchner,  ibid.,  33; 
Buchner,  ibid.,  33;  Albert,  ibid. ,  33;  Albert,  Buchner,  and  Rapp,  ibid.,  3.">;  in  regard 
to  the  opposed  views  see  Macfadyen,  Morris  and  Rowland,  QrieL,  33;  Wroblewski, 
Centralbl.  f.  Physiol.,  13,  and  Journ.  f.  prakt.  Chem.  (N.  F.),  04. 

2  E.  Buchner  and  Meisenheimer,  Ber.  d.  d.  chem.  Gesellsch.,  30;  Herzog,  Zeitschr. 
f.  physiol.  Chem.,  37. 


12  INTRODUCTION. 

cannot  for  the  present  state  anything  positive  in  regard  to  the  importance 
of  these  and  other  intracellular  enzymes  in  the  physiological  processes  in 
the  living  cells.  The  abundant  occurrence  of  oxidases  and  other  enzymes 
of  different  kinds  in  the  cells,  the  increase  in  the  liver  autolysis  found  by 
Jacoby  in  phosphorus  poisoning,  the  solution  of  the  pneumonic  infiltra- 
tion by  autolysis  observed  by  Muller,  and  several  other  observations  l 
seem  to  make  it  probable  that  the  intracellular  enzymes  play  an  impor- 
tant role  in  life,  and  these  enzymes  have  been  considered  as  the  chemical 
tools  of  the  cells. 

Thus  far  no  enzyme  has  been  prepared  in  a  pure  state  with  positiveness 
and  hence  the  nature  of  the  enzymes  and  their  elementary  composition 
is  still  unknown.  The  enzymes  are  considered  as  proteid  bodies  by  many 
investigators,  but  this  opinion  has  not  sufficient  foundation,  and  is  dis- 
puted at  least  for  certain  enzymes.  It  is  indeed  true  that  the  enzymes 
isolated  by  certain  investigators  act  like  genuine  proteid  bodies;  but  it  is 
undecided  whether  or  not  the  products  isolated  in  these  instances  were 
pure  enzymes  or  were  composed  of  enzymes  contaminated  with  proteids. 

The  enzymes  may  be  extracted  from  the  cells  and  tissues  by  means  oi 
water  of  glycerine,  especially  by  the  latter,  which  forms  very  stable  solu- 
tions and  hence  Is  extensively  used  as  a  means  of  extracting  them.  The 
enzymes,  generally  speaking,  do  not  appear  to  be  diffusible,  and  Bredig  2 
has  given  several  reasons,  which  will  be  given  later,  for  considering  them 
not  as  true  solutions  but  rather  colloidal  ones.  The  enzymes  are  also 
absorbed  by  other  colloids  and  are  carried  down  by  fine  precipitates,  and 
this  property  Is  extensively  taken  advantage  of  in  their  preparation.3  The 
enzymes  are  precipitated  from  their  solutions  by  alcohol.  The  continued 
heating  of  their  solutions  above  80°  C.  generally  destroys  most  of  the 
enzymes.  In  the  dry  state,  however,  certain  enzymes  may  be  heated  to 
100°  or  indeed  to  150°-160°  C.  without  losing  their  activity. 

The  action  of  the  enzymes  may  be  markedly  influenced  by  external 
conditions.  The  reaction  of  the  liquid  is  of  special  importance.  Cer- 
tain enzymes  act  only  in  acid,  others,  and  the  majority,  on  the  contrary, 
act  only  in  neutral  or  alkaline  liquids.  Certain  of  them  act  in  very  faintly 
acid  as  well  as  in  neutral  or  alkaline  solutions,  but  best  at  a  specific  reac- 
tion. The  temperature  exercises  also  a  very  important  influence.  In 
general  the  activity  of  enzymes  increases  to  a  certain  limit  with  the  tem- 
perature. This  optimum  is  not  always  the  same,  but,  as  shown  by  Tam- 
mann,  depends,  like  the  destructive  action  of  high  temperatures,  essen- 

1  A  complete  summary  of  the  literature  of  intracellular  enzymes  and  autolysis 
may  be  found  in  Jacoby,  "  Uber  die  Bedeutung  der  intrazelluliiren  Fermente,  etc.," 
Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1.,  1902. 

2  Anorganische  Fermente,  Leipzig,  1901. 

8  See  Briicke,  Wien.  Sitzungsber.,  43,  1861. 


ENZYMES.  13 

tially  upon  the  quantity  of  enzyme  and  other  conditions.  The  products 
of  the  enzymotic  processes  exercise  a  retarding  influence  in  proportion  as 
they  accumulate,  and  indeed  the  enzymotic  process  may  thereby  be 
entirely  stopped.  In  such  cases  of  "false  equilibrium"  (Bredig)  we 
may.  as  shown  by  Tammaxx,1  often  start  the  reaction  again  by  remov- 
ing the  products  of  the  reaction,  by  diluting  with  water,  by  raising 
the  temperature,  by  the  addition  of  more  substance,  or  by  the  addition  of 
more  of  the  enzyme.  The  addition  of  neutral  salts  and  other  substances  of 
various  kinds  may  partly  have  an  accelerating  and  partly  a  retarding  action.2 

We  have  no  characteristic  reactions  for  the  enzymes  in  general,  and 
each  enzyme  is  characterized  by  its  specific  action  and  by  the  conditions 
under  which  it  operates.  According  to  these  actions  most  of  the  enzymes 
studied  can  be  divided  into  two  chief  groups,  namely,  the  hydrolytic  and 
the  oxidizin/i  />pj>ym*s.  The  most  important  subgroups  of  the  hydrolytic 
enzymes  for  animal  life  are  the  proteolytiCj  or  those  which  dissolve  proteid, 
the  lipolytic,  or  fat-splitting,  and  the  atniiloli/ticJ_or  diastatic)  enzymes,  which 
act  upon  the  starches.  To  this  last  group  we  must  include  the  invertases, 
which  split  the  disaccharides  into  monosaccharides.  Among  the  hydro- 
lytic enzymes  we  must  also  include  the  urea-splitting  and  the  glucoside- 
splitting,  the  latter  occurring  in  the  higher  plants.  The  proteid-coagulating 
enzymes,  chymosin  or  casein-coagulating  and  thrombin  or  blood-coagulat- 
ing enzyme,  belong  to  a  special  though  not  clearly  defined  group. 

The  various  oxidases  have  already  been  discussed  in  the  preceding 
pages.  According  to  the  observations  of  certain  investigators  3  we  also 
have  enzymes  which  have  a  reducing  action,  so-called  reductases  or  hydro- 
genases.  To  this  group  belongs  the  so-called  ''philothion''  which  de- 
velops sulphuretted  hydrogen  in  the  presence  of  sulphur  and  water, 
while  other  investigators  have  not  been  able  to  substantiate  this  fact.* 
The  property  of  many  enzymes,  besides  their  specific  action,  of  decompos- 
ing hydrogen  peroxide,  does  not  belong  to  the  enzymes  themselves,  but 
depends  upon  their  contamination  with  another  enzyme  which  has  been 
called  catalase.5 

In  order  to  obtain  a  clear  and  concise  nomenclature  of  the  enzymes  v.  Lipp- 
manx  8  has  suggested  to  construct  the  name  of  the  enzyme  out  of  two  words,  one 

1  The  work  of  Tammann  may  be  found  in  Zeitschr.  f.  physiol.  Chem.,  16,  and 
Zeitschr.  f.  prakt.  Chem.,  3  and  18. 

:  See  Fenni  and  Pernossi,  Zeitschr.  f.  Hygiene,  IS;  also  in  regard  to  the  enzymes 
in  general  see  C.  Oppenheimer,  Die  Fermente,  1900. 

s  See  Abelous  et  Gerard,  Compt.  rend.,  129;   Pozzi-Escot,  Bull.  Soc.  Chim.  (3),  27. 

4  De  Rey-Pailhade,  Recherches  exper.  sur  le  Philothion,  etc.,  Paris,  1S91,  and 
Tsouvelles  recherches  sur  le  Philothion,  Paris,  1S92;  Pozzi-Escot,  1.  c. ;  Chodat  and 
Bach,  Ber.  d.  d.  chem.  Gesellsch.,  30;  Abelous  et  Ribaut,  Compt.  rend.,  137. 

5  See  Al.  Schmidt,  Zur  Blutlehre,  Leipzig,  1S92;  Jacobson,  Zeitschr.  f.  physiol. 
Chem.,  16;  O.  Loew,  foot-note  5,  page  6. 

e  Ber.  d.  d.  chem.  Gesellsch. .  36. 


14  INTRODUCTION. 

of  which  represents  the  substance  acted  upon  by  the  enzyme,  while  the  second 
is  the  important  or  chief  product  produced  by  the  enzyme.  Thus  maltoglucase 
is  an  enzyme  which  produces  J-glucose  from  maltose,  amylmaltase,  one  that 
forms  maltose  from  starch  (amylum),  etc. 

The  action  of  the  enzymes  is  specific,  as  one  and  the  same  enzyme  only 
acts  upon  one  substance  or  a  few  certain  substances  or  groups  of  them. 
Their  action  seems  to  be  entirely  dependent  upon  the  stereometric  con- 
struction of  the  substance  acted  upon,  and  we  can  admit  that  the  enzyme 
attacks  only  specially  arranged  stereometric  atomic  groups,  where  the 
enzyme  fits  the  substance  in  a  maimer  similar  to  a  key  fitting  a  lock 
(E.  Fischer).  E.  Fischer  1  has  given  a  positive  proof  for  the  great 
importance  of  a  different  stereometric  configuration  by  his  investigations 
upon  the  artificially  prepared  series  of  stereoisomeric  glucosides  which  he 
calls  a  and  /?  glucosides.  The  enzymes  of  yeast  infusions  only  act  upon 
the  glucosides  of  the  a-series,  while  emulsin,  on  the  contrary,  only  acts 
upon  those  of  the  ^-series. 

The  best-known  and  most  carefully  studied  enzyme  actions,  the  hydrop- 
ses, are  exothermal  processes,  and  therefore  the  sum  of  the  new  products 
produced  have  a  lower  heat  of  combustion  than  the  original  substance. 
Now,  as  syntheses  are  generally  endothermal  reactioas,  i.e.,  are  processes  re- 
quiring a  taking  up  of  heat  where  external  energy  must  be  supplied  before 
they  take  place,  and  also  as  the  enzymes  are  not  a  source  of  energy,  it 
used  to  be  generally  considered  that  the  enzymes  could  not  bring  about 
any  syntheses.  This  view  is  nevertheless  untenable,  and  it  has  also  been 
shown  that  enzymotic  hydrolyses  may  be  reversible  processes  which  pro- 
duce syntheses.  Croft  Hill  has  shown  that  maltase,  which,  as  is  well 
known,  has  a  splitting  action  upon  maltose,  also  has  the  power  of  regener- 
ating from  glucose  two  isomeric  bioses,  one  a  new  body  called  revertose  and 
another  which  is  probably  maltose  (see  also  Emmerling  2).  Hanriot,3 
Kastle  and  Loevenhardt  4  have  shown  that  the  lipases  can  bring 
about  syntheses,  and  finally  Emmerling5  has  been  able  to  synthesize 
amygdalin  from  mandelic  acidnitrilglucoside,  and  glucose  by  means  of 
the  yeast  maltase.  According  to  Abelous  and  Ribaut  6  the  pig  and 
horse  kidneys  contain  an  enzyme,  which  produces  hippuric  acid  from  benzyl 
alcohol  and  glycocoll.  These  investigators  are  of  the  opinion  that  the 
benzyl  alcohol  is  first  oxidized  to  benzoic  acid  and  then  that  the  synthesis 
is  brought  about  by  the  aid  of  the  energy  set  free  in  this  process.     Them 

1  Zeitschr.  f.  physiol.  Chem.,  26. 

2  Hill,  Ber.  d.  d.  chem.  Gesellsch.,  34,  and  Transactions  Chem.  Society,  1903,  83; 
Emmerling,  Ber.  d.  d.  chem.  Gesellsch.,  34. 

3Compt.  rend.,  132. 

4  The  Amer.  Chem.  Journ.,  24. 

5  Ber.  d.  d.  chem.  Gesellsch.,  34,  3810. 

•Compt.  rend.  Soc.  biol.,  52;  Maly's  Jahresber.,  30. 


ENZYME  ACTION.  15 

is  more  and  moro  tendency  t<»  accept  the  view  that  the  intracellular  enzymes 
are  of  importance  for  the  syntheses  in  the  animal  body. 

The  kind  and  manner  of  the  action  of  enzymes  i-  .-till  unknown.  We 
are  sure  that  they  do  not  occur  among  the  final  products  of  the  reaction; 
still  it  is  qtiite  possible  that  preliminarily  a  transitory  combination  of  the 
enzyme  and  tlie  substance  takes  place,  a  view  which  has  received  consider- 
able support  by  the  work  of  Hanriot  upon  lipase  anil  especially  by  the 
studies  of  Henri  *  on  invertase,  diastase,  and  emulsin.  Certain  investi- 
gations carried  on  during  the  last  few  years,  showing  the  marked  corre- 
spondence  beween  catalysis  and  enzyme  action,  have  been  of  special  impor- 
tance for  a  deeper  insight  into  the  manner  of  enzyme  action.  The  cataly- 
sators,  like  the  enzymes  or  their  derivatives,  are  not  found  in  the  final  prod- 
ucts of  the  reaction,  and  the  quantity  of  the  active  substance  proportionate 
to  the  quantity  of  substance  transformed  is  infinitesimally  small  in  enzyme 
action  as  wpII  «.<*  in  catalysis  In  enzyme  action  as  well  as  in  catalysis  the 
reaction  velocity  seems  to  be  independent  of  the  quantity  of  the  active 
substance  added,  and  this  indicates  that  the  enzyme  action  is  not  to  be 
considered  as  a  starting  of  a  reaction  which  would  not  of  itself  take  place, 
but  rather  as  an  acceleration  of  a  slow,  often  not  noticeable,  proceeding 
chemical  change.  According  to  this  conception  enzyme  action  comes  in 
a  line  with  catalysis,  as,  according  to  Ostwald,2  bodies  are  called  cataly- 
sators  which  by  their  presence  cause  a  change  in  the  reaction  velocity  of 
chemical  processes,  and  indeed  positive  or  negative,  according  as  they 
produce  acceleration  or  retardation.  The  striking  correspondence  between 
enzymes  and  inorganic  catalysators  has  been  shown  especially  by  Bredig 
and  his  collaborators,  v.  Bemek,  Ikeda,  and  Reinders,3  by  their  very 
important   investigations. 

Bredig  has  been  able  to  prepare  colloidal  solutions  of  platinum,  gold, 
and  silver  by  allowing  the  electric  arc  to  play  between  two  poles  of  the 
respective  metal  beneath  water.  These  solutions  of  colloidal  metals, 
metallic  soles,  show  by  their  activity  and  the  dependence  of  this  activity 
upon  external  influences,  even  by  poison,  such  strong  resemblance  to  the 
enzymes  that  Bredig  has  indeed  called  them  inorganic  ferments. 

Still  it  is  nevertheless  true  that  the  manner  of  action  of  catalvsators 
has  not  been  explained,  and  we  must  be  careful  not  to  draw  too  positive 
conclusions  from  the  remarkable  correspondence  of  the  maimer  of  action 
of  metallic  soles  and  certain  ferments.4     This  comparison  between  enzymes 

1  Hanriot,  Compt.  rend.,  132;  Henri,  Lois  gen<5rales  de  Taction  des  diastases, 
Paris,  1903. 

2  Grundriss  d.  allgemein.  Chemie,  3.  Aufl.,  1899. 

'  See  Bredig,  Anorganische  Fermente,  Leipzig,  1901,  and  also  Bredig,  Die  Elements 
d.  chemisehen  Kinetik,  etc.,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  1902. 
4  See  Kastle  and  Loevenhart,  Amer.  Chem.  Jour.,  29. 


16  INTRODUCTION. 

and  catalysators  opens  up  new  lines  of  study  of  enzyme  action  which 
undoubtedly  will  be  very  fruitful. 

As  above  stated,  the  enzymes  are  of  great  importance  for  the  chemical 
processes  going  on  in  the  digestive  tract,  but  we  have  to  add  that  the 
results  of  their  action  are  greatly  complicated  by  processes  of  putrefaction 
which  take  place  in  the  intestine  at  the  same  time,  and  which  are  caused 
by  micro-organisms.  Micro-organisms  therefore  exercise  a  certain  influ- 
ence on  the  physiological  processes  of  the  animal  body.  These  organisms, 
when  they  enter  the  animal  fluids  and  tissues  and  develop  and  multiply, 
are  of  the  greatest  pathological  importance,  and  modern  bacteriology  in 
relation  to  the  doctrine  of  infectious  diseases,  founded  by  Pasteur  and 
Koch,  gives  important  testimony  to  these  facts. 

The  products  produced  by  micro-organisms  may  be  of  very  different 
kinds.  Among  the  substances  produced  in  the  decomposition  of  animal 
fluids  and  tissues  by  putrefactive  organisms  we  find  substances  having  a 
basic  nature.  To  this  class  belong  the  cadaver  alkaloids  called  ptomaines, 
first  found  by  Selmi  in  human  cadavers  and  then  specially  studied  by 
Brieger  and  Gautier.1  Certain  of  these  are  poisonous,  designated  as  tox- 
ins, while  the  others  are  non-poisonous.  They  all  belong  to  the  aliphatic 
compounds  and  generally  do  not  contain  oxygen.  As  an  example  of  these 
basic  substances  we  must  mention  the  two  diamines,  cadaverin  or  pen- 
tamethylendiamine,  C5H14N2,  and  putrescin  or  tetramethylendiamine, 
C4H12N2,  which  have  awakened  special  interest  because  they  occur  in  the 
contents  of  the  intestine  and  in  the  urine  in  certain  pathological  condi- 
tions, especially  in  cholera  and  cystinuria.2 

As  above  stated,  the  chemical  processes  in  animals  and  plants  do  not 
stand  in  opposition  to  each  other;  they  offer  differences  indeed,  but  still 
they  are  of  the  same  kind  from  a  qualitative  standpoint.  Pfluger  says 
that  there  exists  a  blood-relationship  between  all  living  cells  of  the  animal 
and  vegetable  kingdoms,  and  that  they  originate  from  the  same  root;  and 
if  the  unicellular  plant  organisms  can  decompose  protein  substances  in 
such  a  manner  as  to  produce  poisonous  substances,  why  should  not  the 
animal  body,  which  is  only  a  collection  of  cells,  be  able  to  produce  under 
physiological  conditions  similar  poisonous  substances?  It  has  been  known 
for  a  long  time  that  the  animal  body  possesses  this  ability  to  a  great  extent, 
and  as  well-known  evidence  of  this  ability  we  may  mention  various  nitro- 
genized  extractives  and  poisonous  constituents  of  the  secretions  of  certain 
animals.     Those  substances   of  basic   nature  which   are   incessantly   and 

1  Selmi,  Sulle  ptomaine  od  alcaloidi  cadaverici  e  lore-  importanza  in  tossicologia. 
Bologna,  1878;  Ber.  d.  deutsch.  chem.  Gesellsch.,  11.  Correspond,  by  H.  Schiff; — 
Brieger,  Ueber  Ptomaine,  Parts  1,  2,  and  3.  Berlin,  1885-1886;— A.  Gautier,  Traite' 
de  chimie  appliqu<5e  a  la  physiologie,  2,  1873,  and  Compt.  rend.,  91. 

2  See  Brieger,  Berlin,  klin.  Wochenschr. ,  1887;  Baumann  and  Udransky,  Zeitschr. 
f.  physiol.  Chem.,  13  and  15;  Brieger  and  Stadthagen,  Berlin,  klin.  Wochenschr.,  1889. 


PTOMAINES,   TOXINS,  LEUCOMAINES*  17 

regularly  produced  as  products  of  the  decomposition  of  the  protein  sub- 
stances in  the  living  organism,  and  which  therefore  are  to  be  considered 
as  products  of  the  physiological  metabolism,  have  been  called  leucomaines 
by  Gautier  in  contradistinction  to  the  ptomaines  and  toxins  produced 
by  micro-organisms.  These  bodies,  to  which  belong  several  well-known 
animal  extractives,  were  isolated  by  Gautier  '  from  animal  tissues  such 
as  the  muscles.  The  hitherto  known  leucomaines,  of  which  a  few  are 
poisonous  in  small  amounts,  belong  to  the  choline,  the  uric  acid,  and  the 
creatinine  groups. 

The  leucomaines  are  considered  as  being  of  certain  importance  in  caus- 
ing disease.  It  has  been  contended  that  when  these  bodies  accumulate  on 
account  of  an  incomplete  excretion  or  oxidation  in  the  system,  an  auto- 
intoxication may  be  produced  (Bouchard  and  others  2). 

Of  especially  great  interest  are  the  toxins  which  are  found  in  the  higher 
plants  and  animals,  like  the  jequirity-bean  and  castor-seed,  in  the  poison 
of  snakes  and  spiders,  in  blood-serum,  etc.,  and  particularly  those  produced 
by  pathogenic  micro-organisms  which  have  an  unmistakable  relationship 
to  the  enzymes.  A  closer  study  of  these  various  bodies,  lysins,  agglutinins, 
toxins,  etc.,  as  well  as  of  the  antitoxins  and  theory  of  immunity,  does 
not  lie  within  the  scope  of  this  work,  and  although  the  subject  is  of  the 
greatest  importance,  it  cannot  be  treated  here.  We  can  only  call  atten- 
tion to  one  similarity  between  many  toxins  and  enzymes,  and  this  is 
important  in  connection  with  what  we  have  already  stated  in  regard  to 
the  enzymes.  As  by  the  repeated  introduction  of  a  toxin  into  an  animal 
body  we  can  excite  a  formation  of  the  corresponding  antitoxin,  so,  as  first 
shown  by  Morgenroth,3  it  is  also  possible  by  the  increasing  introduction 
of  an  enzyme  (rennin,  for  example)  to  produce  an  antienzyme  (an  antiren- 
nin  )  in  the  body.  This  is  only  a  special  case  of  the  general  immunity  theory 
where  the  animal  body  has  the  power  of  making  foreign  substances  non- 
destructive by  reaction  products  produced  by  the  body. 

1  Bull.  soc.  chim.,  43,  and  A.  Gautier,  Sur  les  alcaloides  derives  de  la  destruction 
bacterienne  ou  physiologique  des  tissus  animaux.     Paris,  1886. 

2  Bouchard,  Lecons  sur  les  auto-intoxications  dans  les  maladies.  Paris,  1887.  See 
also  the  various  text-books  of  clinical  medicine. 

3  Centralbl.  f.  Bacterid,  u.  Parisitenkunde,  26. 


CHAPTER  II. 
THE  PROTEIN  SUBSTANCES. 

The  chief  mass  of  the  organic  constituents  of  animal  tissues  consists  of 
amorphous,  nitrogenized,  very  complex  bodies  of  high  molecular  weight. 
These  bodies,  which  are  either  proteids  in  a  special  sense  or  bodies  nearly 
related  thereto,  take  first  rank  among  the  organic  constituents  of  the  ani- 
mal body  on  account  of  their  great  abundance.  For  this  reason  they  are 
classed  together  in  a  special  group  which  has  received  the  name  'protein 
group  (from  7tpaoT€vo,  I  am  the  first,  or  take  the  first  place) .  The  bodies 
belonging  to  these  several  groups  are  called  protein  substances,  although  in 
a  few  cases  the  proteid  bodies  in  a  special  sense  are  designated  by  the 
same  name. 

The  several  protein  substances1  contain  carbon,  hydrogen,  nitrogen^&nd 
oxygen.  The  majority  nnntain  also  sulphur,  a  few  phosvhoru§.  and  a  few 
also  iron.  Copper,  chlorine,  iodine,  and  bromine  have  been  found  in  some 
few  cases.  On  heating  the  protein  substances  they  gradually  decompose, 
producing  a  strong  odor  of  burnt  horn  or  wool.  At  the  same  time  they 
produce  inflammable  gases,  water,  carbon  dioxide,  ammonia,  nitrogenized 
bases,  besides  many  other  substances,  and  leave  a  large  quantity  of  carbon. 
On  hydrolytic  cleavage  they  all  yield,  besides  nitrogenous  basic  substances, 
especially  large  amounts  of  monamino  acids  of  different  kinds. 

The  nitrogen  occurs  in  the  protein  bodies  in  various  forms,  and  this  is 
also  found  in  the  division  of  the  nitrogen  among  the  cleavage  products. 
On  boiling  with  dilute  mineral  acids  we  obtain  (1)  so-called  amid  nitrogen, 
which  is  readily  split  off  as  ammonia ;  (2)  a  guanidine  residue  which  is  com- 
bined with  diaminovalerianic  acid  as  arginin  and  which  has  also  been  called 
the  urea-forming  group;  (3)  basic  nitrogen,  diamino-acid  nitrogen,  which 
is  precipitated  by  phosphotungstic  acid  as  basic  products  (to  which  also 
the  guanidine  residue  of  arginin  belongs);  (4)  monamino-acid  nitrogen; 
and  (5)  the  nitrogen  in  variable  amounts  which  appears  as  humus-like 
melanoidins,  which  seem  only  to  be  of  secondary  formation  as  products  of 
elaboration. 

1  See  "Eiweisskorper,"  Ladenburg's  Handworterbuch  der  Chemie,  3,  534-589, 
which  gives  a  very  complete  summary  of  the  literature  of  protein  substances  up  to 
1885.  The  more  recent  literature  up  to  the  year  1900  may  be  found  in  O.  Cohnheim, 
Chemie  der  Eiweisskorper.     Braunschweig,  1900. 

18 


NITROGEN  IN   THE  PROTEINS.  19 

The  quantitative  division  of  the  total  nitrogen  between  the  above 
five  groups  is  different  for  the  various  protein  substances,  but  cannot  be 
given  with  certainty,  because  of  the  above-mentioned  melanoidin  forma- 
tion and  the  errors  in  the  methods  used.1  The  following  give  at  least  an 
approximate  idea  of  this  division.2  The  loosely  combined  so-called  amid 
nitrogen  seems'to  be  entirely  absent  in  the  protamins.  In  the  gelatines  we 
find  1-2  percent,  and  5-10  per  cent  in  other  animal  protein  substances;  in 
the  plant  glu ten-pro teids  13-20  per  cent  of  the  total  nitrogen  as  amid  nitrogen. 
The  guanidine  nitrogen  may  amount  in  the  protamins  to  22—44  per  cent  of 
the  total  nitrogen,  in  the  histons  12-13  per  cent,  in  the  gelatines  about  8  per 
cent,  and  in  the  other  protein  bodies  about  2-5  per  cent.  As  basic  nitro- 
gen precipitable  by  phosphotungstic  acid  (including  the  guanidine  residue) 
we  find  63-88  per  cent  in  the  protamins,  35-42.5  per  cent  in  the  histons, 
15-25  per  cent  in  the  other  animal  protein  substances,  5-14  per  cent  in 
zein  and  the  gluten  proteid,  and  about  37  per  cent  in  the  plant  globulin. 
The  chief  quantity  of  the  nitrogen,  55-76  per  cent,  occurs,  with  the  excep- 
tion of  the  protamins,  as  the  monamino-acid  groups.  The  results  for 
the  melanoidin  nitrogen  vary  so  considerably  that  they  will  not  be  men- 
tioned. 

From  the  above  results  it  follows  that  the  nitrogen  of  most  protein 
bodies  exists  in  such  combination  that  the  chief  quantity  appears  in 
the  cleavage  products  as  amino  compounds  on  hydrolytic  cleavage  by 
acids.  By  the  action  of  nitrous  acid  upon  proteins  only  a  very  small  part, 
1-2  per  cent,  of  the  nitrogen  is  evolved,3  which  indicates  that  NH2  groups 
exist  only  to  a  slight  extent  in  these  substances.  It  is  also  generally" 
admitted  that  the  amino  groups  occurring  in  the  cleavage  products  exist 
in  the  original  protein  substance  chiefly  as  imino  groups. 

The  sulphur  occurs  in  the  different  protein  bodies  in  very  different 
amounts.  Certain  of  them,  such  as  the  protamins  and  apparently  also 
certain  bacterial  proteids,4  are  free  from  sulphur;  some,  such  as  gela- 
tine and  elastin,  are  very  poor  in  sulphur;  while  others,  especially  horn  sub- 
stances, are  relatively  rich  in  sulphur.  On  hydrolytic  cleavage  with  min- 
eral acids  the  sulphur  of  the  protein  substances  is  regularly,  at  least  in 
part,  split  off  as  cystin  (K.  Morner)  or,  with  bodies  poorer  in  sulphur, 

1  See  the  work  of  Hausmann,  Zeitschr.  f.  physiol.  Chera.,  27  and  29;  Henderson, 
ibid.,  27;   Kossel  and  Kutscher,  ibid.,  30;   Kutscher,  ibid.,  31,  38;   Hart,  ibid.,  33. 

ee  the  works  given  in  foot-note  1  and  Blum,  Zeitschr.  f.  physiol.  Chem.,  30; 
Kossel,  Ber.  d.  d.  chem.  Gesellsch.,  34,  3214;  Hofmeister,  Ergebnisse  der  Physiol., 
Jahrg.  I,  Abt.  1,  759,  which  also  contains  the  literature;  and  Osborne  and  Harris,  Journ. 
Amer.  Chem.  Soc,  2.Y 

3  See  C.  Paal,  Ber.  d.  d.  chem.  Gesellsch.,  29;  H.  Setoff,  ibid.,  1354;  O.  Loew, 
Chemiker  Zeitung,  1896;  and  O.  Nasse,  Pfluger's  Arch.,  6. 

4  See  Nencki  and  Schaffer,  Journ.  f.  prakt.  Chem.  (N.  F.),  20,  and  M.  Nencki, 
Ber.  d.  d.  chem.  Gesellsch.,  17. 


20  THE  PROTEIN  SUBSTANCES. 

as  cystein  (Embden).  From  certain  protein  substances  a-thiolactic  acid 
(Sitter,  Friedmann,  Frankel),  mercaptans  (Rubner),  or  a  body  having 
an  odor  similar  to  ethyl  sulphide  (Drechsel)  have  been  obtained.1 

A  part  of  the  sulphur  separates  as  potassium  or  sodium  sulphide  on 
boiling  with  caustic  potash  or  soda,  and  may  be  detected  by  lead  acetate 
and  quantitatively  determined  (Fleitmann,  D  anile  wsky,  Kruger,  Fr. 
Schulz,  Osborne,  K.  Morner  2).  What  remains  can  only  be  detected 
after  fusing  with  nitre  and  sodium  carbonate  and  testing  for  sulphates. 
The  relationship  between  the  sulphur  split  off  by  alkali  and  that  not  split 
off  is  different  in  various  proteids.  No  conclusions  can  be  drawn  from 
this  in  regard  to  the  number  of  forms  of  combination  which  the  sulphur 
has  in  the  protein  molecule.  As  shown  by  K.  Morner,  only  about  three- 
fourths  of  the  sulphur  in  cystin  can  be  split  off  by  alkali,  and  the  same  is 
true  for  the  cystin-yielding  complex  of-  the  protein  substances.  If  the 
quantity  of  lead-blackening  sulphur  in  a  protein  body  be  multiplied  by 
f ,  we  obtain  the  quantity  corresponding  to  the  cystin-sulphur  in  the  body. 
By  such  calculation  Morner  found  in  certain  bodies,  such  as  horn  substance, 
seralbumin  and  serglobulin,  that  the  quantity  of  cystin  sulphur  and  total 
sulphur  were  identical,  and  therefore  we  have  no  reason  for  considering 
the  sulphur  in  these  bodies  as  existing  in  more  than  one  form  of  combina- 
tion. In  other  proteins,  such  as  fibrinogen  and  ovalbumin,  on  the  con- 
trary, only  one-half  or  one-third  of  the  sulphur  appeared  as  cystin  sulphur. 

The  constitution  of  the  protein  bodies  is  still  unknown,  although  the 
great  advances  made  in  the  last  few  years  have  brought  us  essentially 
closer  to  the  elucidation  of  the  question.  In  studying  the  constitution  of 
the  protein  bodies  they  have  been  broken  up  in  various  ways  into  simpler 
portions,  and  the  methods  used  for  this  purpose  have  been  of  different 
kinds.  In  these  decompositions  where  the  proteids  in  the  true  sense  have 
been  used,  because  they  can  be  prepared  in  the  crystalline  form,  first 
large  atomic  complexes,  proteoses,  and  peptones  are  obtained  which  still 
have  proteid  characteristics,  and  which  then  suffer  further  decomposition 
until  finally  we  obtain  simpler,  generally  crystalline,  or  at  least  character- 
istic end  products. 

On  heating  proteid  with  barium  hydrate  and  water  in  sealed  tubes  to 
150-250°  C.  Schutzenberger  3  obtained  a  mixture  of  products  among 
which  were  ammonia,  carbon  dioxide,  oxalic  acid,  acetic  acid,  and,  as  chief 
product,  a  mixture  of  amino  acids.      The  conclusion  he  drew  from  this 

1  K.  Morner,  Zeitschr.  f.  physiol.  Chem.,  28,  34;  Embden,  ibid.,  32;  Suter,  ibid., 
20;  Friedmann,  Hofmeister's  Beitrage,  3;  Rubner,  Arch.  f.  Hygiene,  19;  Drechsel, 
Centralbl.  f.  Physiol.,  10,  529;   Frankel,  Sitz.  Ber.  d.  Wien.  Akad.,  112,  II  b,  1903. 

''Fleitmann,  Annal.  der  Chem.  und  Pharm.,  66;  Danilewsky,  Zeitschr.  f.  physiol. 
Chem.,  7;  Kruger,  Pfliiger's  Archiv,  43;  F.  Schulz,  Zeitschr.  f.  physiol.  Chem.,  25; 
Osborne,  Connecticut  Agric.  Expt.  Station  Report  1900;   Morner,  1.  c. 

3  Annal.  de  Chim.  et  Phys.  (5),  16,  and  Bull.  soc.  chim.,  23  and  24. 


DECOMPOSITION  OF  THE  PROTEINS.  21 

experiment,  that  the  proteid  is  a  complex  ureid  or  oxamid,  cannot  be 
considered  for  several  reasons.1 

On  fusing  protcids  with  caustic  alkali  we  obtain  ammonia,  methyl  mer- 
captan,  and  other  volatile  products;  also  leucin,  from  which  then  volatile  fatty- 
acids,  such  as  acetic  acid,  valerianic  acid,  and  also  butyric  acid  are  obtained, 
and  also  tyrosin;  from  which  latter  phenol,  indol,  and  skatol  are  produced- 
As  to  the  products  prepared  by  hydrolytic  cleavage  with  mineral  acids  we 
have  a  number  of  investigations  by  various  experimenters,  especially 
Hlasiwetz  and  Habermann,  Ritthausen  and  Kreusler,  E.  Schulze 
and  his  collaborators,  Drechsel,  Siegfried,  R.  Cohn,  Kossel  and  his 
pupils,  K.  Morner,  and  recently  E.  Fischer  and  his  collaborators.2  The 
chief  products  thus  obtained  are  monamino  acids,  such  as  glycocoll,  alanin, 
aminovalerianic  acid,  leucin,  serin,  tyrosin,  phenylaminopropionic  acid, 
aspartic  and  glutamic  acids,  cystein  and  its  sulphide  cystin;  the  so-called 
hexon  bases,  lysin,  arginin,  and  histidin,  of  which  the  first  two  are  diamino 
acids;  pyrrolidin  and  oxypyrrolidin  carbonic  acids;  sulphuretted  hydrogen, 
ethyl  sulphide,  leucinimide,  ammonia,  and  melanoidins,3  which  latter  seem 
to  be  secondary  condensation  products. 

The  proteids  can  be  split  into  a  large  number  of  bodies  by  the  proteolytic 
enzymes,  and  these  will  be  presented  later.  In  the  first  place  proteoses  and 
peptones  are  produced,  also  an  abundance  of  monamino  acids  of  different 
kinds,  hexon  bases,  tryptophan  (proteinochromogen),  which  is  a  skatol- 
aminoacid,  and  finally  oxyphenylethylamin,  diamins,  and  a  little  ammonia 
and  other  substances. 

A  great  many  substances  are  produced  in  the  putrefaction  of  proteids. 
First  the  same  bodies  as  are  formed  in  the  decomposition  by  means  of 
proteolytic  enzymes  are  produced,  and  then  a  further  decomposition  occurs 
with  the  formation  of  a  large  number  of  bodies  belonging  in  part  to  the 
aliphatic  and  in  part  to  the  aromatic  and  heterocyclic  series.  Of  the  first 
series  we  have  ammonium  salts  of  volatile  fatty  acids,  such  as  caproic, 
valerianic,  and  butyric  acids,  also  succinic  acid,  carbon  dioxide,  methane, 
hydrogen,  sulphuretted  hydrogen,  methyl  mercaptan,  and  others.  The 
ptomaines  also  belong  to  these  products  and  are  probably  in  part  formed 
by  very  different  chemical  processes  or  even  syntheses. 

E.  Salkowski  divides  the  putrefactive  products  of  the  aromatic  and 
heterocyclic  series  into  three  groups:  (a)  the  phenol  group,  to  which  tyrosin, 
the  aromatic  oxyacids,  phenol,  and  cresol  belong;  (6)  the  phenyl  group, 
including  phenylacetic  acid  and  phenylpropionic  acid;  and  lastly  (c)  the 
indol  group,  which  includes  indol,  skatol,  skatolacetic  acid,  and  skatolcar- 
■ — — ■ . — .*»- . 

'See  Habermann  and  Ehrenfeld,  Zeitschr.  f.  physiol.  Chem.,  30. 

2  In  regard  to  the  literature  see  O.  Cohnheim,  Chemie  der  Eiweisskorper,  Braun- 
schweig, 1900,  and  F.  Hofmeister,  Ergebnisse  der  Physiologie,  1,  Abt.  I,  759,  1902. 

8  See  Samuely,  Hofmeister's  Beitrage,  2. 


22  THE  PROTEIN  SUBSTANCES. 

bonic  acid.  These  various  products  are  formed  during  putrefaction  with 
access  of  air.  Nencki  and  Bovet1  obtained  only  p-oxyphenylpropionic 
acid,  phenylpropionic  acid,  and  skatolacetic  acid  on  the  putrefaction  of 
proteids  by  anaerobic  schizomycetes  in  the  absence  of  oxygen.  These 
three  acids  are  produced  by  the  action  of  nascent  hydrogen  on  the  corre- 
sponding amino  acid,  namely,  tyrosin,  phenylaminopropionic  acid,  and 
skatolaminoacetic  acid,  and  these  three  last-mentioned  amino  acids  exist, 
according  to  Nencki,  preformed  in  the  proteid  molecule. 

By  the  moderate  action  of  chlorine,  bromine,  or  iodine  upon  proteids 
these  halogens  enter  in  more  or  less  firm  bondage  with  the  molecule  (Loew, 
Blum,  Blum  and  Vaubel,  Liebrecht,  Hopkins  and  Brook,  Hofmeister, 
Kurajeff  and  others),  and  according  to  the  method  of  procedure  we  can 
prepare  derivatives  having  various  but  constant  amounts  of  halogens  (Hop- 
kins and  Pinkus)  .  The  proteids  are  so  changed  that  they  do  not  split  off 
sulphur  on  treatment  with  alkali,  nor  do  they  respond  to  Millon's  reaction, 
nor  do  they  yield  tyrosin  as  a  cleavage  product.  This  is  ordinarily  ex- 
plained by  the  supposition  that  a  substitution  of  hydrogen  by  iodine  takes 
place  in  the  aromatic  tyrosin  nucleus;  but  according  to  Oswald  the  hetero- 
proteoses,  which  yield  only  very  little  tyrosin,  take  up  about  the  same 
quantity  of  iodine  as  the  protoproteoses,  which  yield  considerable  tyrosin. 
It  seems  as  if  the  iodine  was  united  to  other  groups  besides  the  tyrosin- 
yielding  atomic  complex.  By  the  action  of  iodine  an  oxidation  also  occurs, 
and  Schmidt  2  has  shown  that  a  continuous  splitting  off  of  amino  groups 
takes  place.  According  to  him  phenol  and  p-cresol,  cleavage  products  of 
tyrosin,  besides  benzoic  acid,  are  produced  by  the  oxidation  of  phenylamino- 
propionic acid. 

By  the  oxidation  of  proteid  by  means  of  potassium  permanganate  Maly  3  ob- 
tained an  acid,  oxyprotosulphonic  acid,  C  51.21,  H  6.89,  N  14.59,  S  1.77,  O  25.54 
per  cent,  which  is  not  a  cleavage  product,  but  an  oxidation  product  in  which  the 
group  SH  is  changed  into  SO,  ■  OH.  This  acid  does  not  give  the  proper  color  reac- 
tion with  Millon's  reagent,  yields  no  tyrosin  or  indol,  but  gives  benzene  on 
fusing  with  alkali.  On  continuous  oxidation  Maly  obtained  another  acid,  per- 
oxyproteic  acid,  which  gives  the  biuret  reaction,  but  is  not  precipitated  by  most 
proteid  precipitants.  The  oxy protein' obtained  by  Schulz4  on  the  oxidation 
of  proteid  by  hydrogen  peroxide  is  closely  related   to  oxyprotosulphonic  acid 

1  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  12,  215,  and  27,  302;  Nencki  and  Bovet, 
Monatshefte  f.  Chem.,  10. 

2  Loew,  Journ.  f.  prakt.  Chem.  (N.  F.),  31;  Blum,  Munch,  med.  Wochenschr., 
1896;  Blum  and  Vaubel,  Journ.  f.  prakt.  Chem.  (N.  F.),  57;  Liebrecht,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  30;  Hopkins  and  Brook,  Journ.  of  Physiol.,  22;  Hopkins  and  Pin- 
kus, Ber.  d.  deutsch.  chem.  Gesellsch.,  31;  Hofmeister,  Zeitschr.  f.  physiol.  Chem., 
24;  Kurajeff,  ibid.,  26;  Oswald,  Hofmeister's  Beitrage,  3;  C.  H.  L.  Schmidt,  Zeitschr. 
f.  physiol.  Chem.,  35,  36,  37. 

3  Sitzungsber.  d.  k.  Akad.  d.  Wissensch.  Wien,  91  and  97.  Also  Monatshefte  f. 
Chem.,  6  and  9.  See  also  Bondzynski  and  Zoja,  Zeitschr.  f.  physiol.  Chem.,  19; 
Bernert,  Urid.,  26. 

4  Zeitschr.  f.  physiol.  Chem.,  29. 


CARBOHYDRATE  GROUP  IN   THE  PROTEINS.  23 

in  composition  and  general  characteristics,  but  contains  lead-blackening  sulphur 
and  gives  Mii.i.on's  reaction.  The  oxyprotein  is  claimed  to  be  B  pure  oxida- 
tion product,  while  in  the  production  of  oxyprotosulphonic  acid  Schulz  claims 
that  a  cleavage  takes  place.  On  the  oxidation  of  gelatine  by  ferrous  sulphate 
and  hydrogen  peroxide  Blumbnthal  and  Neuberg1  have  obtained  acetone  as  a 
product.  JOLLBS1  claims  to  have  obtained  large  quantities  of  urea  in  the  oxida- 
tion of  various  proteids  by  potassium  permanganate  in  acid  solution,  but  this 
has  been  disputed  by  other  investigators.  On  the  oxidation  of  protcid  in  acid 
liquids  volatile  fatty  acids,  their  aldehydes,  nitrites  and  ketones,  also  hydrocy- 
anic acid,  benzoic  acid,  and  other  bodies,  have  been  obtained. 

Nitric  acid  gives  various  nitro  products.  A  melanoidin  substance,  xantho- 
melanin,  has  been  obtained  by  v.  Furth.3  Habermann  and  Ehrenfeld  * 
also  obtained  oxyglutaric  acid  among  other  products.  By  the  action  of  bromine 
under  strong  pressure  a  number  of  products  have  been  obtained :  bromanil  and 
tribromacetic  acid,  bromoform,  leucinimid,  leucin,  oxalic  acid,  tribromamino- 
benzoic  acid,  and  other  bodies.  With  aqua  regia,  fumaric  acid,  oxalic  acid,  chor- 
azol,  and  other  bodies  are  obtained.  The  recent  investigations  of  Habf.rmann  and 
Ehrenfeld  and  Panzer  5  upon  the  action  of  chlorine  upon  proteids  and  closely 
related  products  are  important. 

By  the  dry  distillation  of  proteids  we  obtain  a  large  number  of  decomposition 
products  having  a  disagreeable  burnt  odor,  and  a  porous  glistening  mass  of  carbon- 
containing  nitrogen  is  left  as  a  residue.  The  products  of  distillation  are  partly 
an  alkaline  liquid  which  contains  ammonium  carbonate  and  acetate,  ammonium 
sulphide,  ammonium  cyanide,  an  inflammable  oil  and  other  bodies,  and  a  brown 
oil  which  contains  hydrocarbons,  nitrogenized  bases  belonging  to  the  aniline  and 
pyridine  series,  and  a  number  of  unknown  substances. 

The  occurrence  of  protein  substances  which  contain  a  carbohydrate 
group  in  a  glucoside-like  combination  has  been  known  for  some  time. 
The  nature  of  this  carbohydrate,  which  can  be  split  off  by  acid  and  which 
may  amount  to  as  much  as  35  per  cent,  has  been  explained  chiefly  by  the 
investigations  of  Friedrich  Muller  6  and  his  students.  They  have  shown 
that  it  is  always  an  amino  sugar  and  generally  glucosamine.  That  also 
so-called  true  proteids  yield  a  carbohydrate  on  hydrolytic  cleavage  was 
first  shown  by  Pavy,  using  ovalbumin.  The  continued  investigations 
of  Fr.  Muller,  Weydemann,  Skk.maxx,  Fraxkel,  Hofmeisteb,  and 
Langstein  7  have  demonstrated  that  in  these  cases  the  carbohydrate  is  also 
glucosamine.  A  carbohydrate  eomplex,  although  sometimes  only  to  a  very 
slight  amount,  has  also  been  dptectod  in  other  proteids,  ovoglobulin,  serglob- 
ulin,  seralbumin,  pea-globulin,  albumin  of  the  graminese,  y^k^pipleiLl,  and 
fibrin.     In  other  proteids.  on  the  contrary,  such  as  edestin  (of  the  hemp- 

1  Deutsch.  med.  Wochenschr.,  1901. 

*  Zeitschr.  f.  physiol.  Chem.,  32  and  38. 
8  See  Maly's  Jahresber.,  30,  24. 

*  Zeitschr.  f.  physiol.  Chem.,  85. 

5  Habermann  and  Ehrenfeld,  Zeitschr.  f.  physiol.  Chem.,  32;  Panzer,  ibid.,  33 
and  34. 

'  Muller,  Sitzber.  d.  Ges.  d.  Naturw.  zu  Marburg,  1896  and  1S98,  and  Zeitschr.  f. 
Biologie,  42. 

7  In  regard  to  the  literature  on  this  subject  see  the  work  of  Fr.  Muller,  Zeitschr. 
f.  Biologie,  42,  and  Langstein,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  63. 


24  THE  PROTEIN  SUBSTANCES. 

seed)  and  casein,  myosin,  pure  fibrinogen,  and  ovovitellin,  carbohydrates 
have  been  sought  for  with  negative  results.1  All  proteids  hence  do  not 
contain  a  carbohydrate  group,  and  future  investigators  must  therefore 
decide  whether  the  carbohydrate  groups  belong  positively  to  the  proteid 
complex  or  whether  they  are  only  united  with  the  proteid  as  impurities. 
Several  observations  2  show  that  in  working  with  crystalline  proteids  a. 
contamination  with  other  protein  substances  is  unfortunately  not  excluded,, 
and  this  must  not  be  lost  sight  of,  especially  as  the  quantity  of  carbohy- 
drates obtained,  is  often  very  small.  In  the  present  state  of  our  knowl- 
edge we  are  not  warranted  in  considering  the  carbohydrate  groups  as 
belonging  to  the  carbon  nucleus  produced  on  the  destruction  of  the  real 
proteid  complex. 

The  previously  mentioned  methods  used  in  studying  the  structure 
of  the  protein  substances  are  not  of  the  same  value,  but  they  in  part 
substantiate  each  other.  Of  these  we  must  mention  the  hydrolysis  by 
means  of  boiling,  dilute  mineral  acids,  or  by  proteolytic  enzymes,  as  the 
best  methods  for  obtaining  the  carbon  nuclei  in  the  protein  molecule.. 
The  most  important  of  the  carbon  nuclei  obtained  are  as  follows: 

I.  The  Nuclei  belonging  to  the  Aliphatic  Series. 

A.  Sulphur  free,  but  containing  nitrogen:  1.  A  guanidine  residue  (combined 
with  ornithin  as  arginin).  2.  Monobasic  monamino  acids:  Glycocoll  (aminoacetic 
acid),  alanin  (aminopropionic  acid),  aminovalerianic  acid,  leucin  (isobutylamino- 
acetic  acid),  serin  (oxyaminopropionic  acid).  3.  Bibasic  monamino  acids: 
Aspartic  acid  (aminosuccinic  acid)  and  glutamic  acid  (aminoglutaric  acid). 
4.  Monobasic  diamino  acids:  Diaminoacetic  acid  (?),  ornithin  (diaminovalerianic 
acid)  and  lysin  (diaminocaproic  acid).  In  this  group  we  also  include  histidin, 
which  seems  to  be  an  aminocarbonic  acid  of  a  pyrimidine  derivative. 

B.  Sulphurized:  Cystein  (aminothiolactic  acid)  and  its  sulphide  cystin  (diam- 
inodithiodilactylic  acid),  thiolactic  acid,  mercaptans,  and  ethyl  sulphide. 

II.  The  Nuclei  belonging  to  the  Carbocyclic  Series. 
Phenylaminopropionic  acid  and  tyrosin. 

III.   The   Nuclei  belonging  to  the  Heterocyclic  Series. 

A.  Of  the  pyrrol  group:  Pyrrolidin  carbonic  acid  and  oxypyrrolidin  carbonic 
acid. 

B.  Of  the  indol  group:  Tryptophan,  or  skatolaminoacetic  acid  (skatol- 
acetic  acid,  skatolcarbonic  acid,  indol,  and  skatol  in  putrefaction).3 

In  regard  to  these  carbon  nuclei  it  must  be  remarked  that  they  are 
not  all  found  in  every  protein  body  thus  far  investigated,  and  also  that 
the  one  and  the  same  cleavage  product,  such,  for  example,  as  glycocoll, 
leucin,  tyrosin  or  cystin,  is  obtained  in  very  variable  amounts  from  differ- 
ent protein  substances.  It  is  very  difficult  to  say  to  what  extent  all 
the  above-mentioned  carbon  nuclei  exist  in  the  protein  molecule.      Still 

1  See  foot-note  7,  page  23. 

-  See  Wichmann,  Zeitschr.  f.  physiol.  Chem.,  23,  and  N.  Schulz,  Die  Grosse  des- 
Eiweissmolekuls,  Jena,  1903,  51. 

According  to  certain  observations  a  pyridine  nucleus  also  exists  in  the  proteid. 
See  Loew,  Journ.  f.  prakt.  Chem.,  31;   Samuely,  Hofmeister's  Beitriige,  2. 


SYNTHESIS  OF  THE  PROTEINS.  25 

the  fact  is  not  excluded  that  in  the  hydrolysis  certain  carbon  nuclei  may 
be  secondarily  formed  from  others.  We  cannot  exclude  the  possibility, 
as  suggested  by  Loew,1  that  in  the  hydrolysis  a  marked  atomic  displace- 
ment perhaps  occurs  before  cleavage,  and  for  this  reason  two  carbon  nuclei, 
such  as  leuein  and  lysin  or  tyrosin  and  phenylalanin,  may  be  produced 
from  the  same" atomic  groupings,  each  according  to  the  nature  of  the 
neighboring  groups. 

Even  if  we  admit  the  above,  still  jt  is  undoubtedly  true  that  the 
chief  cleavage  products  of  the  protein  substances  are  amino  acids.  As 
Hofmeister,2  from  chemical  coasiderations,  has  explained,  we  can  also 
consider  the  proteids  as  chiefly  formed  by  condensation  of  ammo  acids 
where  the  amino  acids  are  united  to  each  other  by  means  of  mono 
groups  according;  to  the  following  scheme; 

— NH.CH.CO— NH.CH.CO NH.CH.CO— NH.CH.CO— 

C4H9  CH2.C6H,(OH)    CH2.COOH    C3H0.CH2.NH2 

(Leuein)  (Tyrosin)  (Aspartic  acid)  (Lysin) 

Closely  connected  with  the  above  is  the  question  as  to  how  far  is  it 
possible  to  prepare  proteid-like  substances  synthetically.  In  this  con- 
nection we  must  mention  that  Grimaux  and  then  also  Schutzenberger 
and  Pickering  have  been  able  to  prepare  substances  which  in  many  prop- 
erties are  similar  to  the  proteids  from  various  amino  acids  either  alone 
or  mixed  with  other  bodies  such  as  biuret,  alloxan,  xanthine,  or  ammo- 
nia. Of  much  greater  interest  is  the  chaining  together  of  amino  acids, 
as  performed  by  Curtius  and  especially  by  E.  Fischer.3  This  last  inves- 
tigator has  succeeded  in  preparing  complex  bodies  called  by  him  di-  or 
polypeptides  by  uniting  two  or  more  amino  acid  groups.  For  example, 
glycylglycin  and  glycylalanin  anhydride  are  dipeptides.  As  an  example 
of  the  polypeptides  we  must  mention  the  carbethoxyldiglycylleucin  ester 
and  the  carbethoxyltriglycylglycin  ester,  in  which  4  glycin  molecules  are 
united  in  anhydride  form.  Several  of  these  synthetically  prepared  bodies 
give  the  biuret  reaction,  and  they  may  be  considered  as  the  beginning  of  a 
proteid  synthesis. 

It  is  at  present  impossible  to  decide  on  a  proper  classification  of  the  protein 
substances.  A  grouping  based  upon  their  chemical  constitution  is  not  pos- 
sible, and  their  general  properties,  solubilities,  and  precipitation  properties 

1  Loew,  Die  chem.  Energie  d.  lebenden  Zellen,  Miinchen,  1898,  and  Hofmeister's 
Beitriige,  1. 

2"t"ber  den  Bau  des  Eiweissmolekiils."  Gesellsch.  Deutsch.  Xaturforscher  und 
Artze,  Verhandl.  1902,  and  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  759. 

3  See  Pickering,  King's  College,  London,  Physiol.  Lab.  Collect.  Papers,  1S97,  which 
also  cites  Grimaux's  work;  also  Journ.  of  Physiol.,  IS,  and  Proceed.  Roy.  Soc,  60, 
1897;  Schutzenberger,  Compt.  rend.,  106  and  112;  Curtius,  Journ.  f.  prakt.  Chem., 
26;   E.  Fischer,  Ber.  d.  d.  chem.  Gesellsch.,  35  and  36. 


26  THE  PROTEIN  SUBSTANCES. 

are  too  uncertain  to  aid  us  in  this  connection.  On  the  other  hand  a  classifi- 
cation is  important,  and  we  cannot  do  without  one,  so  we  will  give  the  follow- 
ing systematic  summary  of  the  chief  groups  of  the  protein  bodies  as  suggested 
by  Hoppe-Seyler  and  Drechsel,  which  will  be  of  some  aid  to  us. 

I.  Simple  Proteids  or  Albuminous  Bodies. 

.  i  Seralbumin, 

|  Lactalbumin,  and  others. 

c  Fibrinogen, 

Globulins ■]  Myosin, 

(  Serglobulins,  and  others. 

.  ( Casein, 

Nucleoalbumins -J  ~      .  '  .  ,     ,, 

( Ovovitellin,  and  others. 

.  „    .    .  (  Acid  albuminate, 

Albuminates \ ■  .  „   , .    „  ' 

( Alkali  albuminate. 

Proteoses  (and  Peptones). 

8  ' "  *  '  (  Proteids  coagulated  by  heat,  and  others. 

(Protamins  and  Histons). 

II.  Compound   Proteids. 

Haemoglobins. 

(  Mucins  and  Mucinoids, 

Glucoproteids <  Amyloid, 

L  Ichthulin,  and  others. 

_T     ,           .  . «  ( Nucleohiston, 

Nucleoproteids -|  „  ±    7  ,  .  ,     ,, 

(  Cytoglobm,  and  others. 

III.  Albumoids  or  Albuminoids. 

Keratins. 

Elastin. 

Collagen. 

Reticulin. 

(Fibroin,  Sericin,  Cornein,  Spongin,  Conchiolin,  Byssus,  and  others.) 
To  this  summary  must  be  added  that  we  often  find  in  the  investigations 
of  animal  fluids  and  tissues  protein  substances  which  do  not  coincide  with 
the  above  scheme,  or  do  so  only  with  difficulty.  At  the  same  time  it  must 
be  remarked  that  bodies  will  be  found  which  seem  to  rank  between  the 
different  groups,  hence  it  is  very  difficult  to  sharply  divide  these  groups. 

I.  Simple  Proteids  or  Albuminous  Bodies. 

The  simple  proteids  are  nevBr-faj^qg  constituents  of  the  animal  and 
vegetable  organisms .__  They  are  especially  found  in  the  animal  body,  where 
they  form  the  solid  constituents  of  the  muscles,  and  the  blood-serum,  and 


SIMPLE  P  ROTE  IDS.  27 

they  are  so  generally  distributed  that  there  are  only  a  few  animal  w ■< n- 
tions  and  excretions,  such  as  the  tears,  perspiration,  and  perhaps  urine, 
in  which  they  are  entirely  ahsent  or  only  occur  as  traces. 

All  proteids  contain  carbon,  hydrogen,  nitrogen,  oxygen,  and  sulphur:1 
a  few  contain  also  phosphorus.  Iron  is  generally  found  in  traces  in  their 
ash,  and  it  seems  to  be  a  regular  constituent  of  a  certain  group  of  the 
albuminous  bodies,  namely,  the  nucleoalbumins^  The  composition  of 
the  different  albuminous  bodies  varies  a  little,  but  the  variations  are  within 
relatively  close  limits.  For  the  better  studied  animal  proteids  the  follow- 
ing composition  of  the  ash-free  substance  has  been  given: 

C 50.6  —54.5    per  cent. 

H 6.5  —  7.3 

N 15.0  —17.6 

S 0.3—2.2 

P 0.42—  0.85 

0 21.50—23.50 

The  animal  proteids  are  odorless,  tasteless,  and  ordinarily  amorphous. 
The  crystalloid  spherules  (Dotterplattchen)  occurring  in  the  eggs  of  certain 
fishes  and  amphibians  do  not  consist  of  pure  proteids,  but  of  proteids 
containing  large  amounts  of  lecithin,  which  seem  to  be  combined  with 
mineral  substances.  Crystalline  proteids  2  have  been  prepared  from  the 
seeds  of  various  plants,  and  lately  crystallized  animal  proteids  (see  seral- 
bumin and  ovalbumin,  Chapters  VI  and  XIII)  have  also  been  prepared. 
In  the  dry  condition  the  proteids  appear  as  white  powders,  or  when  in 
thin  layers  as  yellowish,  hard,  transparent  plates.  A  few  are  soluble* 
in  water,  others  only  soluble  in  salt  or  faintly  alkaline  or  acid  solutions, 
while  others  are  insoluble  in  these  solvents.  All  proteids  when  burnt 
leave  an  ash,  and  it  is  therefore  questionable  whether  there  exists  any 
proteid  body  which  is  soluble  in  water  without  the  aid  of  mineral  sub- 
stances. Nevertheless  it  has  not  been  thus  far  successfully  proved  that 
a  native  proteid  body  can  be  prepared  perfectly  free  from  mineral  sub- 
stances without  changing  its  constitution  or  its  properties.3 

The  proteids  do  not  diffuse  through  animal  membranes  or  do  so  only 
to  a  very  slight  extent,  and  hence  have  in  most  cases  a  pronounced  col- 
loidal nature  in  Graham's  sense.  As  colloids  they  can,  like  other  pro- 
tein substances,  to  a  more  or  less  degree,  prevent  the  precipitation  of  a 

1  See  foot-note  4,  page  19. 

'See  Maschke,  Journ.  f.  prakt.  Chem.,  74;  Drechsel,  ibid.  (N.  F.),  19;  Griibler, 
tbid.  (X.  F.),  23;  Ritthausen,  ibid.  (N.  F.),  25;  Schmiedeberg,  Zeitschr.  f.  physioL 
Chem.,  1;   Weyl,  ibid.,  1. 

3  See  E.  Harnack,  Ber.  d.  d.  chem.  Gesellsch.,  22,  23,  25,  and  31;  Werigo,  Pfluger's 
Archiv,  4S;  Bulow,  ibid.,  5Sj  Schulz,  Die  Grosse  des  Eiweissmolekuls,  Jena,  1903. 


28  THE  PROTEIN  SUBSTANCES. 

colloidal  metallic  solution  (gold  solution)  by  means  of  an  electrolyte  (see 
gold  equivalent  according  to  Zsigmondy  and  Schulz).1  The  proteids 
are  optically  active  and  rotate  the  plane  of  polarization  to  the  left. 

Although  certain  of  the  proteids,  i.e.  casein,  have  an  acid  character, 
others  on  the  contrary,  like  the  histons,  have  a  more  pronounced  basic 
character;  still  the  proteids  may  be  considered  as  amphoteric  electrolytes, 
i.e.  they  may  functionate  as  weak  acids  as  well  as  weak  baseband  they 
yield  salts  which  are  strongly  hydrolytically  dissociated.  The  acid-com- 
bining power  of  various  proteids  is  different  and  the  maximum  acid-com- 
bining power  may  perhaps  also  be  used  in  the  differentiation  of  the  vari- 
ous proteids  (Cohnheim,  Eeb,  and  others). 

The  acid-combining  power  of  the  proteids  has  been  studied  according  to 
physical  methods  by  Sjoquist,  Bugarsky,  and  Liebermann  and  by  chemical 
methods  by  Spiro  and  Pemsel,  Erb,  Cohnheim  and  Krieger,  v.  Rhorer. 
The  methods  pursued  by  Cohnheim  and  Krieger  consisted  in  precipitating 
the  proteid  from  acid  solution  (HC1)  with  an  alkaloid  reagent  (calcium  phos- 
photungstate).  The  reaction  took  place  as  follows:  proteid  hydrochloride+ 
calcium  phosphotungstate  =  proteid  phosphotungstate +  calcium  chloride.  The 
acid  remaining  in  the  filtrate  was  determined,  and  when  this  quantity  was  sub- 
tracted from  the  known  original  amount  in  the  proteid  solution,  the  difference 
represented  the  acid  combined  with  the  proteid.  If  sodium  picrate  or  potassium- 
mercuric  iodide  is  used  instead  of  the  phosphotungstate  we  have,  according  to 
v.  Rhorer,2  a  method  which  is  the  best  of  all  heretofore  suggested. 

The  proteids  can  be  salted  out  from  their  neutral  solution  by  neutral 
salts  (NaCl,  Na2S04,  MgS04,  (NH4)2S04,  and  many  others)  in  sufficient 
concentrations.  While  by  other  precipitants  they  are  often  changed  or 
modified,  their  properties  remain  unchanged  on  salting  out  and  the  proc- 
ess is  reversible,  as  on  diminishing  the  concentration  of  the  salt  the  pre- 
cipitate redissolves.  The  various  proteids  act  essentially  different  towards 
the  same  salt,  which  is  of  the  greatest  importance  in  the  separation  of 
the  proteids.3  For  one  and  the  same  proteid  the  behavior  towards  differ- 
ent neutral  salts  is  different,  as  some  cause  a  precipitate,  while  others 
on  the  contrary  do  not  precipitate. 

According  to  Pauli  4  this  can  be  explained  by  the  fact  that  we  have  to  do 
with  ion  action  and  that  the  precipitation  action  is  the  algebraic  sum  of  the  antago- 
nistic properties.  If  we  ascribe  the  precipitating  action  to  the  cations  and  a 
retarding  action  upon  precipitation  to  the  anions,  then,  according  as  a  salt  has 
the  positive  cations  or  the  negative  anions  in  excess,  we  obtain  a  precipitation 
action  or  not  or  it  is  accelerated  or  retarded. 

Those  proteids  which  occur,  according  to  the  common  views,  preformed  in 
the  animal  fluids  and  tissues  and  which  have  been  isolated  from  these  by 

1  Hofmeister's  Beitriige,  3. 

2  Pfiiiger's  Arch.,  90.  In  regard  to  the  literature  on  this  subject  see  Cohnheim, 
Chemie  der  Eiweisskorper,  pages  22  and  27. 

8  See  Cohnheim,  1.  c,  page  12;    Pinkus,  Journ.  of  Physiol.,  27;    Pauli,  Hofmeister's 
Beitriige,  3,  225;   Spiro,  Hofmeister's  Beitriige,  4. 
4  Pauli,  Hofmeister's  Beitriige,  3. 


PRECIPITATION  REACTIONS  OF   THE  PRO TE IDS.  29 

indifferent  chemical  means  without  loosing  their  original  properties  are 
called  native  proteids  New  modifications  having  other  properties—can 
be  obtained  from  the  native  proteids  by  hqatfogj  ^Y  f^p  FPtion  of  various 
chemical  reagents  such  as  acids,  alkalies,  alcohol.  an4  others,  as  well  as  by 
proteolytic  enzymes.  These  new  proteids  are  called  modified  ("  denatu- 
rierte")  proteids,  to  differentiate  them  from  the  native  proteids.  In  the 
scheme  given  on  page  26  the  albumins,  globulins,  and  nucleoalbumins 
belong  to  the  native  proteids  and  the  acid  or  alkali  albuminates,  the  pro- 
teoses, the  peptones,  and  the  coagulated  proteid  to  the  modified  proteids. 

On  heating  a  solution  of  a  native  proteid  it  is  modified  at  various 
temperatures,  depending  upon  the  different  proteid  present.  With  proper 
reaction  and  other  favorable  conditions,  for  instance  in  the  presence  of 
neutral  salts,  most  proteids  can  in  this  way  be  precipitated  in  a  solid  form 
as  coagulated  proteid.  The  various  temperatures  at  which  a  coagula- 
tion of  different  proteids  occurs  in  neutral  solutions  containing  salt,  have 
in  many  cases  given  us  good  means  for  detecting  and  separating  several 
proteids.  The  views  in  regard  to  the  use  of  these  means  are  somewhat 
divided.1 

A  modification  can  be  brought  about  also  by  the  action  of  acids,  alka- 
lies, or  salts  of  the  heavy  metals,  in  certain  cases  by  water  alone,  also  by 
the  action  of  alcohol,  chloroform,2  and  ether  and  by  violent  shaking,  etc. 

The  general  reactions  for  the  proteids  are  very  numerous,  but  only  the 
most  important  will  be  given  here.  To  facilitate  the  study  of  these  they 
have  been  divided  into  the  two  following  groups: 

A.  Precipitation  Reactions  of  the  Proteid  Bodies. 

1.  Coagulation  Test.  An  alkaline  proteid  solution  does  not  coagulate 
on  boiling,  a  neutral  solution  only  partly  and  incompletely,  and  the  reaction 
must  therefore  be  acid  for  coagulation.  The  neutral  liquid  is  first  boiled 
and  then  the  proper  amount  of  acid  added  carefully.  A  flocculent  precipi- 
tatc  is  formed,  and  if  properly  done  the  filtrate  should  be  water-clear.  If 
dilute  acetic  acid  be  used  for  this  test,  the  liquid  must  first  be  boiled  and 
then  1,  2,  or  3  drops  of  acid  added  to  each  10-15  c.c,  depending  on  the 
amount  of  proteid  present,  and  boiled  before  the  addition  of  each  drop.  If 
dilute  nitric  acid  be  used,  then  to  10-15  c.c.  of  the  previously  boiled  liquid 

1  See  Halliburton,  Journ.  of  Physiol.,  5  and  11;  Corin  and  Berard,  Bull,  de  l'Acad. 
roy.  de  Belg.,  15;  Haycraft  and  Duggan,  Brit.  Med.  Journ.,  1890,  and  Proc.  Roy. 
Soc.  Edin.,  1889;  Corin  and  Ansiaux,  Bull,  de  l'Acad.  roy.  de  Belg.,  21;  L.  Fredericq, 
Centralbl.  f.  Physiol.,  3;  Haycraft,  ibid.,  4;  Hewlett,  Journ.  of  Physiol.,  13;  Duclaux, 
Annal.  Institut  Pasteur,'".  In  regard  to  the  relationship  of  the  neutral  salts  to  the 
heat  coagulation  of  albumins  see  also  Starke,  Sitzungsber.  d.  Gesellsch.  f.  Morph.  u. 
Physiol,  in  Miinchen,  1897;    Pauli,  Pfliiger's  Arch.,  78. 

'See  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  31;  Fr.  Kriiger,  Zeitschr.  f.  Biologie, 
41;  Loew  and  Aso,  Bull.  Coll.  Agric.  Tokio,  4. 


30  THE  PROTEIN  SUBSTANCES. 

15-20  drops  of  the  acid  must  be  added.  If  too  little  nitric  acid  be  added,  a 
soluble  combination  of  the  acid  and  proteid  is  formed,  which  is  precipitated 
by  more  acid.  A  proteid  solution  containing  a  small  amount  of  salts  must 
first  be  treated  with  about  1  per  cent  NaCl,  since  the  heating  test  may  fail, 
especially  on  using  acetic  acid,  in  the  presence  of  only  a  slight  amount  of 
proteid.  2.  Behavior  towards  Mineral  Acids  at  Ordinary  Temperatures. 
The  proteids  are  precipitated  by  the  three  ordinary  mineral  acids  and  by 
metaphosphoric  acid,  but  not  by  orthophosphoric  acid.  If  nitric  acid  be 
placed  in  a  test-tube  and  the  proteid  solution  be  allowed  to  flow  gently 
thereon,  a  white  opaque  ring  of  precipitated  proteid  will  form  where  the 
two  liquids  meet  (Heller's  albumin  test).  3.  Precipitation  by  Metallic 
Salts.  Copper  sulphate,  neutral  and  basic  lead  acetate  (in  small  amounts), 
mercuric  chloride,  and  other  salts  precipitate  proteid.  On  this  is  based  the 
use  of  proteids  as  antidotes  in  poisoning  by  metallic  salts.  4.  Precipitation 
by  Ferro-  or  Ferricyanide  of  Potassium  in  Acetic-acid  Solution.  In  these 
tests  the  relative  quantities  of  reagent,  proteid,  or  acid  do  not  interfere  with 
the  delicacy  of  the  test.  5.  Precipitation  by  Neutral  Salts,  such  as  Na2S04 
or  NaCl,  when  added  to  saturation  to  the  liquid  acidified  with  acetic  acid 
or  hydrochloric  acid.  6.  Precipitation  by  Alcohol.  The  solution  must  not 
be  alkaline,  but  must  be  either  neutral  or  faintly  acid.  It  must,  at  the 
same  time,  contain  a  sufficient  quantity  of  neutral  salts,  (jj  Precipitation 
by  Tannic  Acid  in  acetic-acid  solutions.  The  absence  of  neutral  salts  or  the 
presence  of  free  mineral  acids  may  prevent  the  appearance  of  the  precipitate, 
but  after  the  addition  of  a  sufficient  quantity  of  sodium  acetate  the  precipi- 
tate will  in  both  cases  appear.  8.  Precipitation  by  Phosphotungstic  or 
Phosphomolybdic  Acids  in  the  presence  of  free  mineral  acids.  Potassium- 
mercuric  iodide  and  potassium-bismuth  iodide  precipitate  proteid  solutions 
acidified  with  hydrochloric  acid.  9.  Precipitation  by  Picric  Acid  in  solu- 
tions acidified  by  organic  acids.  10.  Precipitation  by  Trichloracetic  Acid 
in  2-5  per  cent  solutions,  and  11,  by  Salicylsulphonic  Acid.  The  proteids  are 
precipitated  by  nucleic  acid,  taurocholic  and  chondroitin-sulphuric  acid  in 
acid  solutions. 

B.  Color  Reactions  for  Proteid  Bodies. 

1,  Millon's  Reaction.1  A  solution  of  mercury  in  nitric  acid  containing 
some  nitrous  acid  gives  a  precipitate  with  proteid  solutions  which  at  the 
ordinary  temperature  is  slowly,  but  at  the  boiling-point  more  quickly, 
colored  red;  and  the  solution  may  also  be  colored  a  feeble  or  bright 
red.     Solid  albuminous  bodies,  when  treated  by  this  reagent,  give  the  same 

1  The  reagent  is  obtained  in  the  following  way :  1  pt.  mercury  is  dissolved  in  2  pts. 
nitric  acid  (of  sp.  gr.  1.42),  first  cold  and  then  warmed.  After  complete  solution  of 
the  mercury  add  1  volume  of  the  solution  to  2  volumes  of  water.  Allow  this  to  stand 
a  few  hours  and  decant  the  supernatant  liquid. 


COLOR  REACTIONS  OF   THE  PSOTBIDS.  31 

coloration.  This  reaction,  which  depends  on  the  presence  of  the  aromatic 
group  in  the  proteid,  is  also  given  by  tyrosin  and  other  monohydroxyl 
benzene  derivatives.  According  to  O.  Nasse  1  it  is  best  to  use  a  solution 
of  mercuric  acetate  which  is  treated  with  a  few  drops  of  a  1  per  cent 
solution  of  potassium  or  sodium  nitrite;  previous  to  use  a  few  drops 
of  acetic  acid  are  added.  (^Xanthoproteic  Reaction.  With  strong^  nitric 
acid  the  albuminous  bodies  give,  on  heating  to  boiling,  yellow  flakes 
or  a  yellow  solution.  After  saturating  with  ammonia  or  alkalies  the  color 
becomes  orange-yellow.  (§)  Adamkiewicz's  Reaction.  If  a  little  proteid 
is  added  to  a  mixture  of  1  vol.  concentrated  sulphuric  acid  and  2  vols. 
glacial  acetic  acid  a  reddish-violet  color  is  obtained  slowly  at  ordinary  tem- 
peratures, but  more  quickly  on  heating.  According  to  Hopkins  and  Cole  a 
this  reaction  only  takes  place  in  using  glacial  acetic  acid  containing  gly- 
oxylic  acid.  According  to  them  it  is  better  to  use  a  solution  of  glyoxylic 
acid.  A  dilute  aqueous  solution  of  the  acid  or  some  of  the  solid  acid  is 
added  to  the  proteid  solution  and  sulphuric  acid  allowed  to  flow  down  the 
side  of  the  test-tube,  when  the  reddish-violet  color  will  appear  at  the  point  of 
contact  of  the  two  liquids.  Gelatine  does  not  give  this  reaction.  (Q  Biuret, 
test._  If.  a  proteid  solution  be  first  treated  with  caustic  potash  or  soda 
and  then  a  dilute  copper  sulphate  solution  be  added  drop  by  drop,  first 
a  reddish,  then  a  reddish-violet,  and  lastly  a  violet-blue  color  is  obtained. 
5.  Proteids  are  soluble  on  heating  with  concentrated  hydrochloric  arid,  pro- 
ducing a  violet  color,  and  when  they  are  previously  boiled  with  alcohol 
and  then  washed  with  ether  (Liebermann  3)  they  give  a  beautiful  blue 
solution.  6.  With  concentrated  sulphuric  acid  and  sugar  (in  small  quanti- 
ties) the  albuminous  bodies  give  a  beautiful  red  coloration.4 

Many  of  these  color  reactions  are  obtained  as  shown  by  Salkowski  5  by  the 
aromatic  or  heterocyclic  cleavage  products  of  the  proteids.  Millon's  reaction 
is  only  obtained  by  the  substances  of  the  phenol  group;  the  Xanthoproteic 
reaction  by  the  phenol  group  and  skatol  or  skatolcarbonic  acid.  Liebermann's 
reaction  is  not  given  by  any  of  the  aromatic  splitting  products.  Adamkiewicz's 
reaction  is  only  given  by  the  indol  group,  especially  skatolcarbonic  acid.  Lie- 
Hermann's  reaction,  as  well  as  the  reaction  with  sulphuric  acid  and  sugar,  seems 
at  least  to  be  a  furfurol  reaction.  The  biuret  reaction  is  not  only  given  by  pro- 
tein substances  but  also  by  many  other  bodies.  According  to  H.  Schiff  8  this  reac- 
tion occurs  with  those  bodies  containing  two  amino  groups,  CONH2,  CSNHj, 
C(NH)  NH2  or  also  CH2NH,,  united  either  directly  by  their  carbon  atoms  or  by  means 
of  a  third  carbon  or  nitrogen  atom.     As  examples  of  such  bodies  we  can  mention 

1  See  O.  Nasse,  Sitzungsb.  d.  Naturforsch.  Gesellsch.  zu  Halle,  1879,  and  Pfltiger's 
Arch.,  S3;  see  also  Vaubel  and  Blum,  Jo-urn.  f.  prakt.  Chem.  (N.  F.),  57. 

2  Proceed.  Roy.  Sqc,  68. 

3  Centralbl.  f.  d.  med.  Wissensch.,  1887;  see  also  Cole,  Journ.  of  Physiol.,  30. 

4  In  regard  to  the  precipitation  and  coloration  reactions  of  proteids  with  aniline 
dyes  see  M.  Heidenhain,  Pfliiger's  Arch.,  90  and  96. 

5  Zeitschr.  f.  pbysiol.  Chem.,  12. 

8  Ber.  d.  d.  chem.  Gesellsch.,  29  and  30. 


32  THE  PROTEIN  SUBSTANCES. 

several  diamines  or  ami noam ides,  such  as  oxamide,  biuret,  glyeinamide,  a-  and  /?- 
aminobutyramide,  aspartic  acid  amide,  etc.  The  biuret  reaction  alone  is  there- 
fore no  proof  as  to  the  proteid  nature  of  a  substance,  for  example,  urobilin  gives 
a  very  similar  color  reaction,  and  a  protein  substance  can  still  retain  its  protein 
nature,  as  by  the  action  of  nitrous  acid  or  by  a  splitting  off  of  ammonia,  although 
it  does  not  give  the  biuret  reaction. 

The  delicacy  of  the  various  reagents  differs  for  the  different  proteids,  and 
on  this  account  it  is  impossible  to  give  the  degree  of  delicacy  for  each  reac- 
tion for  all  proteids.  Of  the  precipitation  reactions  Heller's  test  (if  we 
eliminate  the  peptones  and  certain  proteoses)  is  recommended  in  the  first 
place  for  its  delicacy,  though  it  is  not  the  most  delicate  reaction,  and  because 
it  can  be  performed  so  easily.  Among  the  precipitation  reactions,  that 
with  basic  lead  acetate  (when  carefully  and  exactly  executed)  and  the 
reactions  6,  7,  8,  9,  and  11  are  the  most  delicate.  The  color  reactions  1  to 
4  show  great  delicacy  in  the  order  in  which  they  are  given. 

Xo  proteid  reaction  is  in  itself  characteristic,  and,  therefore,  in  test- 
ing for  proteids  one  reaction  is  not  sufficient,  but  a  number  of  precipi- 
tation and  color  reactions  must  be  employed. 

For  the  quantitative  estimation  of  coagulable  proteids  the  determina- 
tion by  boiling  with  acetic  acid  can  be  performed  with  advantage,  since,  by 
operating  carefully,  it  gives  exact  results.  Treat  the  proteid  solution  with 
a  1-2  per  cent  common-salt  solution,  or  if  the  solution  contains  large  amounts 
of  proteid  dilute  with  the  proper  quantity  of  the  above  salt  solution,  and 
then  carefully  neutralize  with  acetic  acid.  Now  determine  the  quantity 
of  acetic  acid  necessary  to  completely  precipitate  the  proteids  in  small 
measured  portions  of  the  neutralized  liquid  which  have  previously  been 
heated  on  the  water-bath,  so  that  the  filtrate  does  not  respond  to  Hel- 
ler's test.  Xow  warm  a  larger  weighed  or  measured  quantity  of  the 
liquid  on  the  water-bath,  and  add  gradually  the  required  quantity  of  acetic 
acid,  with  constant  stirring,  and  continue  heating  for  some  time.  Filter, 
wash  with  water,  extract  with  alcohol  and  then  with  ether,  dry,  weigh, 
incinerate,  and  weigh  again.  With  proper  work  the  filtrate  should  not  give 
Heller's  test.  This  method  serves  in  most  cases,  and  especially  so  in 
cases  where  other  bodies  are  to  be  quantitatively  estimated  in  the  filtrate. 

The  precipitation  by  means  of  alcohol  may  also  be  used  in  the  quan- 
titative estimation  of  proteids.  The  liquid  is  first  carefully  neutralized, 
treated  with  some  NaCl  if  necessary,  and  then  alcohol  added  until  the 
solution  contains  70-80  vol.  per  cent  anhydrous  alcohol.  The  precipitate 
is  collected  on  a  filter  after  24  hours,  extracted  with  alcohol  and  ether, 
dried,  weighed,  incinerated,  and  again  weighed.  This  method  is  only 
applicable  to  liquids  which  do  not  contain  any  other  substances,  like  glyco- 
gen, which  are  insoluble  in  alcohol. 

In  both  of  these  methods  small  quantities  of  proteid  may  remain  in  the 
filtrates.  These  traces  may  be  determined  as  follows:  Concentrate  the 
filtrate  sufficiently,  remove  any  separated  fat  by  shaking  with  ether,  and 
then  precipitate  with  tannic  acid.  Approximately  63  per  cent  of  the  tannic 
acid  precipitate,  washed  with  cold  water  and  then  dried,  may  be  considered 
as  proteid. 

In  many  cases  good  results  may  be  obtained  by  precipitating  all  the 


ALBUMINS  AND  GLOBULINS.  33 

protcid  with  tannic  acid  and  determining  the  nitrogen  in  the  washed  pre- 
cipitate by  means  of  Kjeldahl's  method.  On  multiplying  the  quantity 
of  nitrogen  found  by  6.25  we  obtain  the  quantity  of  protcid. 

The  removal  of  proteids  from  a  solution  may  in  most  cases  be  performed 
l>v  boiling  w.th  acetic  acid.  Small  amounts  of  proteid  which  remain  in  the 
filtrates  may  be  separated  by  boiling  with  freshly  precipitated  lead  car- 
bonate or  with  ferric  acetate,  as  described  by  Hofmeister.  If  the  liquid 
cannot  be  boiled,  the  proteid  may  be  precipitated  by  the  very  careful  addi- 
tion of  lead  acetate,  or  by  the  addition  of  alcohol.  If  the  liquid  contains 
substances  which  are  precipitated  by  alcohol,  such  as  glycogen,  then  the 
proteid  may  be  removed  by  the  alternate  addition  of  potassium-mercuric- 
iodide  and  hydrochloric  acid  (see  Chapter  VIII,  on  Glycogen  Estimation), 
or  also  by  trichloracetic  acid  as  suggested  by  Obermayer  and  Frankel.2 

In  the  precipitation  of  proteid  as  well  as  the  quantitative  estimation  by  means 
of  heat,  it  must  be  borne  in  mind,  as  shown  by  Spiro,3  that  several  nitrogenous 
substances,  such  as  piperidine,  pyridine,  urea,  etc.,  disturb  the  coagulation  of  the 
proteids. 

Synopsis  of  the  Most  Important  Properties  of  the  Different  Chief  Groups 

of  Proteids. 

As  it  is  not  possible  to  base  the  classification  of  the  different  proteid 
groups  according  to  their  constitution,  we  are  obliged  to  make  use  of  their 
different  solubilities  and  precipitation  properties  in  their  general  characteriza- 
tion.    As  there  exists  no  sharp  difference  between  the  various  groups  in    • 
this  regard  it  is  impossible  to  drawT  a  sharp  line  between  them. 

Albumins.  These  bodies  are  soluble  in  water  and  are  not  precipitated 
"by  the  addition  of  a  little  acid  or  alkali.  They  are  precipitated  by  the 
addition  of  large  quantities  of  mineral  acids  or  metallic  salts.  Their  solu- 
tion in  water  coagulates  on  boiling  in  the  presence  of  neutral  salts,  but  a 
weak  saline  solution  does  not.  \jl  NaCl  or  MgS04  is  added  to  saturation 
to  a  neutral  solution  in  water  at  the  normal  temperature  or  at  30°  C.  no 
precipitate  is  formed;  but  if  acetic  acid  is  added  to  this  saturated  solution 
the  albumins  readily  separate.  When  ammonium  sulphate  is  added  in 
substance  to  saturation  to  an  albumin  solution  a  complete  precipitation 
occurs  at  ordinary  temperature.  Of  all  the  native  proteids  the  albumins 
are  the  richest  in  sulphur,  containing  from  1.6  per  cent  to  2.2  per  cent. 

Globulins.  These  substances  are  insoluble  in  water,  but  dissolve  in 
dilute  neutral  salt  solutions.  The  globulins  are  precipitated  unchanged 
from  these  solutions  by  sufficient  dilution  with  water,  and  on  heating  they 
coagulate.  The  globulins  dissolve  in  water  on  the  addition  of  very-  little 
acid  or  alkali,  and  on  neutralizing  the  solvent  they  precipitate  again. 

The  solution  in  a  minimum  amount  of  alkali  is  precipitated  by  carbon 

1  Zcitschr.  f.  physiol.  Chem.,  2  and  4. 

•Obermayer,  Wein.  mod.  Jahrbiicher,  1S3S;   Frankel,  Pfliiger's  Arch.,  52  and  oo. 

3  Zeitschr.  f.  physiol.  Chem.,  30. 


34  THE  PROTEIN  SUBSTANCES. 

dioxide,  but  the  precipitate  may  be  redissolved  by  an  excess  of  the  precipi- 
tant. jThe  neutral  solutions  of  the  globulins  containing  salts  are  partly  or 
completely  precipitated  on  saturation  with  NaCl  or  MgS04  in  substance  at 
normal  temperatures.  The  globulins  are  completely  precipitated  by  half- 
saturating  with  ammonium  sulphate^  The  globulins  contain  an  average 
amount  of  sulphur,  generally  not  below  1  per  cent. 

A  sharp  line  cannot  be  drawn  between  the  globulins  and  albumins  as  shown 
by  the  properties  of  the  serglobulins.  The  same  is  true  between  the  globulins 
and  the  albuminates.  Several  globulins  are  very  readily  changed  by  the  action  of 
very  little  acid,  as  also  by  standing  under  water  when  in  a  precipitated  condition, 
into  albuminates,  and  then  become  insoluble  in  neutral  salt  solutions.  Osborne,1 
who  has  closely  studied  this  property  in  connection  with  edestin  (from  hemp- 
seed),  considers  the  globulin,  "globan,"  which  has  been  made  insoluble  in  salt 
solution,  as  an  intermediate  step  in  the  formation  of  the  albuminate  which  is  pro- 
duced by  the  hydrolytic  action  of  the  H  ions  of  water  or  of  the  acid.  According 
to  J.  Starke  2  the  globulins  are  not  soluble  in  dilute  salt  solutions,  but  form  alkali 
proteid  compounds  whose  solubility  in  salts  is  brought  about  by  an  increase  in 
the  free  OH  ions  produced  by  the  salts.  This  view  is  not  tenable  for  several 
globulins  and  seems  in  fact  not  to  be  well  founded. 

Nucleoalbumins.  This  group  of  phosphorized  proteids  is  found  widely 
diffused  in  both  the  animal  and  vegetable  kingdoms.  The  nucleoalbumins 
behave  like  weak  acids ;  they  are  nearly  insoluble  in  water,  but  dissolve 
easily  with  the  aid  of  a  little  alkali.  The  nucleoalbumins  resemble  certain 
of  the  globulins  and  albuminates  in  solubility  and  precipitation  properties, 
but  differ  from  these  two  groups  by  containing  phosphorus.  They  stand 
close  to  the  nucleoproteids  by  their  content  of  phosphorus,  but  differ  from 
these  in  not  yielding  any  purin  bases  on  cleavage.  It  has  not  yet  been 
found  possible  to  obtain  from  the  nucleoalbumins  any  proteid-free  pseudo- 
nucleic  acids  corresponding  to  the  nucleic  acids,  but  only  acids  rich  in 
phosphorus,  which  always  give  the  proteid  reactions  (Levene  and  Alsberg, 
Salkowski  3).  For  this  reason  the  nucleoalbumins  cannot  be  classed  as 
compound  proteids.  In  peptic  digestion  a  proteid  rich  in  phosphorus  can 
be  split  off  from  most  nucleoalbumins,  and  this  has  been  called  para-  or 
pseudonuclein.  The  claim  made  by  Liebermann  that  the  pseudonuclein 
is  a  combination  of  proteid  with  metaphosphoric  acid  has  been  shown  to 
be  incorrect  by  the  investigations  of  Giertz.4  The  nucleoalbumins  always 
seem  to  contain  some  iron. 

1  Zeitschr.  f.  physiol.  Chem.,  33. 

2  Zeitschr.  f.  Biologie,  40  and  42.  In  regard  to  the  various  views  on  this  subject 
see  Wolff  and  Smits,  ibid.,  41;  Osborne,  1.  c. ;  Hammarsten,  Ergebnisse  der  Physiologie, 
1,  Abt.  1;  Moll,  Hofmeister's  Beitrage,  4. 

3  Levene  and  Alsberg,  Zeitschr.  f.  physiol.  Chem.,  31;  Salkowski,  ibid.,  32;  Levene, 
ibid.,  32. 

*  Liebermann,  Ber.  d.  deutsch.  chem.  Gesellsch.,  21;  Giertz,  Zeitschr.  f.  physiol. 
Chem.,  2S 


ALBUMINATES.  35 

The  separation  of  pseudonuclein  in  the  peptic  digestion  of  nucleoalbumins 
cannot  be  considered  as  positively  characteristic  of  the  nucleoalbumin  group. 
The  extent  of  such  a  cleavage  is  dependent  upon  the  intensity  of  the  pepsin  diges- 
tion, the  degree  of  acidity,  and  the  relationship  between  the  nuceloalbumins 
and  the  digestive  fluids.  The  separation  of  a  pseudonuclein  may,  as  shown  by 
Salkowski,  not  occur  even  in  the  digestion  of  ordinary  casein,  and  Wroblewskv 
did  not  obtain  any  pseudonuclein  at  all  in  the  digestion  of  the  casein  from  human 
milk.  Wiman  l"  has  also  shown  in  the  digestion  of  vegetable  nucleoalbumin 
that  the  obtainment  of  considerable  pseudonuclein  or  none  is  dependent  upon  the 
way  in  which  the  digestion  is  performed.  The  most  essential  characteristic  of 
this  group  of  proteids  is  that  they  contain  phosphorus,  and  that  the  xanthine 
bases  are  absent  in  their  cleavage  products. 

fThe  nucleoalbumins  are  often  confounded  with  nucleoproteids  and  also 
with  phosphorized  glucoproteids.  From  the  first  class  they  differ  by  not 
yielding  any  xanthine  bodies  when  boiled  with  acids,  and  from  the  second 
group  by  not  yielding  any  reducing  substance  on  the  same  treatment.        J 

Lecithalbumins.  In  the  preparation  of  certain  protein  substances  products 
are  often  obtained  containing  lecithin,  and  this  lecithin  can  only  be  removed 
with  difficulty  or  incompletely  by  a  mixture  of  alcohol  and  ether.  Ovovitellin 
is  such  a  protein  body  containing  considerable  lecithin,  and  Hoppe-Seyler 
considers  it  a  combination  of  proteid  and  lecithin.  Liebermann  2  has  obtained 
proteids  containing  lecithin  as  an  insoluble  residue  on  the  peptic  digestion  of  the 
mucous  membrane  of  the  stomach,  liver,  kidneys,  lungs,  and  spleen.  He  con- 
siders them  as  combinations  of  proteid  and  lecithin  and  calls  them  lecithalbumins. 
Further  investigation  of  these  bodies  is  desirable. 

Alkali  and  Acid  Albuminates.  The  native  proteids  are  modified  by  the 
action  of  sufficiently  strong  acids  or  alkalies./"  By  the  action  of  alkalies 
all  native  albuminous  bodies  are  converted,  with  the  elimination  of  nitro- 
gen or  by  the  action  of  stronger  alkali,  also  with  the  emission  of  sulphur, 
into  a  new  modification,  called  alkali  albuminate,  whose  specific  rotation 
is  increased  at  the  same  time.  If  caustic  alkali  in  substance  or  in  strong 
solution  be  allowed  to  act  on  a  concentrated  proteid  solution,  such  as 
blood-serum  or  egg-albumin,  the  alkali  albuminate  may  be  obtained  as  a 
solid  jelly  which  dissolves  in  water  on  heatingjand"  which  is  called  "Lieber- 
kuhn's  solid  alkali  albuminate."  By  the  action  of  dilute  caustic  alkali 
solutions  on  dilute  proteid  solutions  we  have  alkali  albuminates  formed 
slowly  at  the  ordinary  temperature,  but  more  rapidly  on  heating.  These 
solutions  may  be  modified  by  the  source  of  the  proteid  acted  upon,  and 
also  by  the  extent  of  the  action  of  the  alkali,  but  still  they  have  certain 
reactions  in  common. 

If  proteid  is  dissolved  in  an  excess  of  concentrated  hydrochloric  acid,  or 
if  we  digest  a  proteid  solution  acidified  with  1-2  p.  m.  hydrochloric  acid  in 
the  thermostat,  or  digest  the  proteid  for  a  short  time  with  pepsin-hydro- 
chloric acid,  we  obtain  new  modifications  of  proteid  which  indeed  may  show 

1  Salkowski,  Pfliiger's  Arch.,  63; — Wr6blewsky,  Beitrage  zur  Kennitniss  des  Frauen- 
kasei'ns.     Inaug.-Diss.  Bern,  1894; — Wiman,  Upsala  Lakaref.  Forh.  N.  F.f  2. 

'Hoppe-Seyler,  Med.  chem.  Untersuch.,  1868;  also  Zeitschr.  f.  physiol.  Chem.,  13, 
479 j  Liebermann,  Pfliiger's  Archiv,  50  and  54. 


36  THE  PROTEIN  SUBSTANCES. 

somewhat  varying  properties,  but  have  certain  reactions  in  common.  These 
modifications,  which  ma}''  be  obtained  in  a  solid  gelatinous  condition  on 
sufficient  concentration,  are  called  acid  albuminates  or  acid  albumins,  and 
sometimes  syntonin,  though  we  prefer  to  apply  the  term  syntonin  to  the 
acid  albuminate,  which  is  obtained  by  extracting  muscles  with  hydrochloric 
acid  of  1  p.  m,J 

The  alkali  and  acid  albuminates  have  the  following  reactions  in  com- 
mon: They  are  nearly  insoluble  in  water  and  dilute  common-salt  solu- 
tion (see  page  34),  but  they  dissolve  readily  in  water  on  the  addition  of  a 
very  small  quantity  of  acid  or  alkali.  Such  a  solution  as  nearly  neutral  as 
possible  does  not  coagulate  on  boiling,  but  is  precipitated  at  the  normal  tem- 
perature on  neutralizing  the  solvent  by  an  alkali  or  an  acid.  A  solution  of 
an  alkali  or  acid  albuminate  in  acid  is  easily  precipitated  on  saturating 
with  NaCl,  but  a  solution  in  alkali  is  precipitated  with  difficulty  or  not  at 
all,  according  to  the  amount  of  alkali  it  contains.  Mineral  acids  in  excess 
precipitate  solutions  of  acid  as  well  as  alkali  albuminates.  The  nearly  neu- 
tral solutions  of  these  bodies  are  also  precipitated  by  many  metallic  salts. 

Notwithstanding  this  agreement  in  the  reactions,  the  acid  and  alkali 
albuminates  are  essentially  different,  for  by  dissolving  an  alkali  albuminate 
in  some  acid  no  acid  albuminate  solution  is  obtained,  nor  is  an  alkali 
albuminate  formed  on  dissolving  an  acid  albuminate  in  water  by  the  aid  of 
a  little  alkali.  In  the  first  case  we  obtain  a  combination  of  the  alkali 
albuminate  and  the  acid  soluble  in  water,  and  in  the  other  case  a  soluble 
combination  of  the  acid  albuminate  with  the  alkali  added.  The  chemical 
process  in  the  modification  of  proteids  with  an  acid  is  essentially  different 
from  the  modification  with  an  alkali,  hence  the  products  are  of  a  different 
kind.  The  alkali  albuminates  are  relatively  strong  acids.  They  may  be 
dissolved  in  water  with  the  aid  of  CaC03,  with  the  elimination  of  C02, 
which  does  not  occur  with  typical  acid  albuminates,  and  they  show  in 
opposition  to  the  acid  albuminates  also  other  variations  which  stand  in 
connection  with  their  strongly  marked  acid  nature.  Dilute  solutions  of 
alkalies  act  more  energetically  on  proteids  than  do  acids  of  corresponding 
concentration,  fin  the  first  case  a  part  of  the  nitrogen,  and  often  also  the 
sulphur,  is  split  off,  and  from  this  property  we  may  obtain  an  alkali  albu- 
minate by  the  action  of  an  alkali  upon  an  acid  albuminate;  but  we  cannot 
obtain  an  acid  albuminate  by  the  reverse  reaction  (K.  Morner  *).  For 
this  reason  the  calling  of  the  modified  proteid  obtained  by  the  action  of 
alkali  or  acid,  protein,  the  combination  of  this  protein  with  alkali,  alkali 
albuminate,  and  the  combination  with  acid,  acid  albuminate,  leads  to  a 
misunderstanding  or  to  a  wrong  conception.    / 

r  The  preparation  of  the  albuminates  has  been  given  above.  The  cor- 
responding albuminate  obtained  by  the  action  of  alkalies  or  acids  upon  a 

1  Pfliiger's  Arch.,  17. 


PROTEOSES  AND  PEPTONES.  37 

proteid  solution  may  be  precipitated  by  neutralizing  with  acid  or  alkali. 
The  washed  precipitate  is  dissolved  in  water  by  the  aid  of  a  little  alkali  or 
acid,  and  again  precipitated  by  neutralizing  the  solvent.  If  this  precipi- 
tate which  has  been  washed  in  water  is  treated  with  alcohol  and  ether, 
the  albuminate  will  be  obtained  in  a  pure  form.  J 

In  the  preparation  of  acid  as  well  as  alkali  albuminates  proteoses  and  the 
nearly  related  albuminates  are  formed.  The  "alkali  albumose"  obtained  by 
Maas  l  belongs  to  this  class.  The  lysalbinic  acid  and  protalbinic  acid  obtained 
by  Paal2  from  ovalbumin  are  likewise  alkali  albuminates.  Desaminoalbuminic 
acid  is  an  alkali  albuminate  which  Schmiedeberg  3  obtained  by  the  action  of  such 
weak  alkali  that  a  part  of  the  nitrogen  was  evolved,  but  the  quantity  of  sulphur 
remained  the  same.  The  proteid  combination  obtained  by  Blum  by  the  action 
of  formol  on  proteid  and  called  by  him  protogen  has  similarities  with  the  alkali 
albuminates  in  regard  to  solubilities  and  precipitation,  but  is  not  identical 
therewith.4 

Proteoses  and  Peptones.  /  Peptones  are  designated  as  the  final  products 
of  the  decomposition  of  proteid  bodies  by  means  of  proteolytic  enzymes, 
in  so  far  as  these  final  products  are  still  true  proteids,  while  we  designate 
as  proteoses  (albumoses,  or  propeptones) ,  the  intermediate  products  pro- 
duced in  the  peptonization  of  proteids  in  so  far  as  they  are  not  sub- 
stances similar  to  albuminates^'  Proteoses  and  peptones  may  also  be  pro- 
duced by  the  hydrolytic  decomposition  of  the  proteids  with  acids  or  alkalies, 
also  by  the  putrefaction  of  the  same.  They  may  also  be  formed  in  very 
small  quantities  as  by-products  in  the  investigations  of  animal  fluids  and 
tissues,  and  the  question  as  to  what  extent  these  exist  preformed  under 
physiological  conditions  requires  very  careful  investigation. 

Between  the  peptone  which  represents  the  final  cleavage  product  and 
the  proteose  which  stand  closest  to  the  original  proteid  we  have  undoubt- 
edly a  series  of  intermediate  products.  Under  such  circumstances  it  is  a 
difficult  problem  to  try  to  draw  a  sharp  line  between  the  peptone  and 
the  proteose  group,  and  it  is  just  as  difficult  to  define  our  conception  of 
peptones  and  proteoses  in  an  exact  and  satisfactory  manner. 

The  proteoses  (or  albumoses)  have  been  considered  as  those  proteid  bodies 
whose  neutral  or  faintly  acid  solutions  do  not  coagulate  on  boiling  and 
which,  to  distinguish  them  from  peptones,  were  characterized  chiefly  by 
the  following  properties.  The  watery  solutions  are  precipitated  at  the 
ordinary  temperature  by  nitric  acid  as  well  as  by  acetic  acid  and  potassium 
ferrocyanide,  and  this  precipitate  has  the  peculiarity  of  disappearing  on 

1  Zeitschr.  f.  physiol.  Chem.,  30. 

1  Ber.  d.  d.  chem.  Gesellsch.,  35. 

s  Arch.  f.  exp.  Path.  u.  Pharm.,  39. 

4  Blum,  Zeitschr.  f.  physiol.  Chem.,  22.  The  older  investigations  of  Loew  may 
be  found  in  Maly's  Jahresber.,  1SS8.  On  the  action  of  formaldehyde  see  also  Benedi- 
centi,  Du  Bois-Reyinond's  Arch.,  1S97;  S.  Schwartz,  Zeitschr.  f.  physiol.  Chem.,  30 j 
Bliss  and  Novy,  Journ.  of  Exper.  Med.,  4. 


38  THE  PROTEIN  SUBSTANCES. 

heating  and  reappearing  on  cooling.  If  a  proteose  solution  is  saturated 
with  NaCl  in  substance,  the  proteose  is  partly  precipitated  in  neutral 
solutions,  but  on  the  addition  of  acid  saturated  with  salt  it  is  more 
completely  precipitated.  This  precipitate,  which  dissolves  on  warming,  is 
a  combination  of  the  proteose  with  the  acid. 

We  formerly  designated  as  peptone  those  proteid  bodies  which  are  readily 
soluble  in  water  and  which  do  not  coagulate  by  heat,"  whose  solutions  are 
precipitated  neither  by  nitric  acid,  nor  by  acetic  acid  and  potassium  ferro- 
cyanide,  nor  by  neutral  salts  and  acid. 

The  reactions  and  properties  which  the  proteoses  and  peptones  have  in 
common  were  formerly  considered  as  the  following :  They  give  all  the  color 
reactions  of  the  proteids,  but  with  the  biuret  test  they  give  a  more  beautiful 
red  color  than  the  ordinary  proteids.  They  are  precipitated  by  ammoniacal 
lead  acetate,  by  mercuric  chloride,  tannic,  phosphotungstic,  phospho- 
molybdic  acids,  potassium-mercuric  iodide  and  hydrochloric  acid,  and  also 
by  picric  acid.  They  are  precipitated  but  not  coagulated  by  alcohol, 
namely,  the  precipitate  obtained  is  soluble  in  water  even  after  being  in 
contact  with  alcohol  for  a  long  time.  The  proteoses  and  peptones  also 
have  a  greater  diffusive  power  than  native  proteids,  and  the  diffusive 
power  is  greater  the  nearer  the  questionable  substance  stands  to  the  final 
product,  the  now  so-called  pure  peptone. 

These  old  views  have  gradually  undergone  an  essential  change.  After 
Heynsius'  1  observation  that  ammonium  sulphate  was  a  general  pre- 
cipitant for  proteids,  also  peptone  in  the  old  sense,  Kuhne  and  his  pupils  2 
proposed  this  salt  as  a  means  of  separating  proteoses  and  peptones.  Those 
products  of  digestion  which  separate  on  saturating  their  solution  with 
ammonium  sulphate  or  can  indeed  be  salted  out  are  considered  by  Kuhne 
and  indeed  by  most  of  the  modern  investigators  as  proteoses,  while  those 
which  remain  in  solution  are  called  peptones  or  pure  peptones.  These 
pure  peptones  are  formed  in  relatively  large  amounts  in  pancreatic  diges- 
tion, while  in  pepsin  digestion  they  are  only  formed  in  small  quantities 
or  after  prolonged  digestion. 

According  to  Schutzenberger  and  Kuhne  3  the  proteids  yield  two 
chief  groups  of  new  proteid  bodies  when  decomposed  by  dilute  mineral 
acids  or  with  proteolytic  enzymes;  of  these  the  anti  group  shows  a  greater 
resistance  to  further  action  of  the  acid  and  enzyme  than  the  other,  namely, 

1  Pfluger's  Archiv,  34. 

2  See  Kuhne,  Verhandl.  d.  naturhistor.  Vereins  zu  Heidelberg  (X.  F.),  3;  J.  "Wenz, 
Zeitschr.  f.  Biologie,  22;  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologie,  22;  R.  Neu- 
meister,  ibid.,  23;  Kiihne,  ibid.,  29. 

3  Schiitzenberger,  Bull,  de  la  soc.  chimique  de  Paris,  23;  Kiihne,  Verhandl.  d. 
naturhist.  Vereins  zu  Heidelberg  (N.  F.),  1  and  Kiihne  and  Chittenden,  Zeitschr.  f. 
Biologie,  19.     See  also  Paal,  Ber.  d.  deutsch.  chem.  Gesellsch.,  27. 


PROTEOSES  AND  PEPTONES.  39 

the  hemi  group.  These  two  groups  are,  according  to  Kuhne,  united  in 
the  different  proteoses,  even  though  in  various  relative  amounts,  and  each 
proteose  contains  the  anti  as  well  as  the  hemi  group.  The  same  is  true  for 
the  peptone  obtained  in  pepsin  digestion,  hence  he  calls  it  amphopcptone. 
In  tryptic  digestion  a  cleavage  of  the  amphopcptone  takes  place  into  anti- 
peptone  and  hcmipeptone.  Of  these  two  peptones  the  hemipeptone  is  further 
split  into  amino  acids  and  other  bodies  while  the  antipeptone  Ls  not  attacked. 
By  the  sufficiently  energetic  action  of  trypsin  only  one  peptone  is  at  last 
obtained,  the  so-called  antipeptone. 

Ki'uxE  and  his  pupils,  who  have  conducted  extensive  investigations 
on  the  proteoses  and  peptones,  classify  the  various  proteoses  according 
to  their  different  solubilities  and  precipitation  properties.  In  the  pepsin 
digestion  of  fibrin  '  they  obtained  the  following  proteoses :  (a)  Hetero- 
protcose,  insoluble  in  water  but  soluble  in  dilute  salt  solution;  (b)  Proto- 
proteose,  soluble  in  salt  solution  and  water.  These  two  proteoses  are 
precipitated  by  NaCl  in  neutral  solutions,  but  not  completely.  Hetero- 
proteose  may,  by  being  in  contact  with  water  for  a  long  time  or  by  drying, 
be  converted  into  a  modification,  called  (c)  Dysproteose,  which  is  insoluble 
in  dilute  salt  solutions,  (d)  Deuteroproteose  is  a  proteose  which  is  soluble 
in  water  and  dilute  salt  solution  and  which  is  incompletely  precipitated 
from  acid  solution  by  saturating  with  NaCl  and  not  precipitated  from 
neutral  solutions.  This  precipitate  is  a  combination  of  the  proteose  with 
acid  (Herth  2).  The  heteroproteose  is  essentially  the  same,  as  described 
by  Brucke,  as  peptone. 

The  proteoses  obtained  from  different  proteid  bodies  do  not  seem  to  be 
identical,  but  differ  in  their  behavior  to  precipitants.  Special  names  have 
been  given  to  these  various  proteoses  according  to  the  mother-proteid, 
namely,  albumoscs,  globuloses,  vitelloses,  caseoses,  myosinoses,  etc.  These 
various  proteoses  are  further  distinguished,  as  proto-,  hetero-,  and  deutero- 
caseoses  for  example.  Chittenden  3  has  suggested  the  common  name 
proteoses  for  the  products  formed  intermediary  between  the  proteids  and 
peptones  in  the  digestion  of  animal  and  vegetable  proteids.  We  have 
made  use  of  it  in  this  sense  in  preference  to  the  word  albumose  (which  is 
used  in  the  German  and  by  some  other  writers),  but  which  will  be  used 
in  this  book  as  indicating  the  intermediary  products  in  the  hydrolysis 
of  albumins  and  not  as  a  general  term.  Certain  proteoses  have  also  been 
obtained  in  a  crystalline  state  (Schrotter)  . 

1  See  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  20. 

7  Monatshefte  f.  Chem.,  5. 

3  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  22  and  25;  Xeumeister,  ibid.,  23; 
Chittenden  and  Hartwell,  Journ.  of  Physiol.,  11  and  12;  Chittenden  and  Painter, 
Studies  from  the  Laboratory,  etc.,  Yale  University,  2,  New  Haven,  1891;  Chittenden, 
ibid.,  3;  Sebelien,  Chem.  Centralblatt,  1890;  Chittenden  and  Goodwin,  Journ.  of 
Physiol.,  12. 


40  THE  PROTEIN  SUBSTANCES. 

Neumeister1  designates  as  atmidalbumose  that  body  which  is  obtained  by 
the  action  of  superheated  steam  on  fibrin.  At  the  same  time  he  also  obtained  a 
substance  called  atmidalbumw ,  which  stands  between  the  albuminates  and  the 
proteoses. 

Of  the  soluble  proteoses  Neumeister  designates  the  protoproteose  and 
heteroproteose  as  'primary  proteoses,  while  the  deuteroproteoses,  which  are 
closely  allied  to  the  peptones,  he  calls  secondary  proteoses.  As  an  essen- 
tial difference  between  the  primary  and  secondary  proteoses  he  suggests  the 
following : 2  The  primary  proteoses  are  precipitated  by  nitric  acid  in  salt- 
free  solutions,  while  the  secondary  proteoses  are  only  precipitated  in  salt 
solutions,  and  certain  deuteroproteoses,  such  as  deuterovitellose  and  deu- 
teromyosinose,  are  only  precipitated  by  nitric  acid  in  solutions  saturated 
with  NaCl.  The  primary  proteoses  are  precipitated  from  neutral  solutions 
by  copper  sulphate  solution  (2  :  100),  also  by  NaCl  in  substance,  while  the 
secondary  proteoses  are  not.  The  primary  proteoses  are  completely  pre- 
cipitated from  their  solution  saturated  with  NaCl  by  the  addition  of  acetic 
acid  saturated  with  salt,  while  the  secondary  proteoses  are  only  partly 
precipitated.  The  primary  proteoses  are  readily  precipitated  by  acetic 
acid  and  potassium  ferrocyanide,  while  the  secondary  are  only  incompletely 
precipitated  after  some  time.  The  primary  proteoses  are  also,  according 
to  Pick,3  completely  precipitated  by  ammonium  sulphate  (added  to  one  half 
saturation),  while  the  secondary  proteoses  remain  in  solution. 

The  true  peptones,  as  they  used  to  be  obtained,  are  exceedingly  hygro- 
scopic, and  when  perfectly  dry  sizzle  like  phosphoric  anhydride  when 
treated  with  a  little  water.  They  are  exceedingly  soluble  in  water,  diffuse 
more  readily  than  the  proteoses,  and  are  not  precipitated  by  ammonium 
sulphate.  In  contradistinction  to  the  proteoses  the  true  peptones  are  not 
precipitated  by  nitric  acid  (even  in  solution  saturated  with  salt),  by  acetic 
acid  saturated  with  salt  and  sodium  chloride,  potassium  ferrocyanide 
and  acetic  acid,  picric  acid,  trichloracetic  acid,  potassium-mercuric  iodide, 
and  hydrochloric  acid.  They  are  precipitated  by  phosphotungstic  acid, 
phosphomolybdic  acid,  corrosive  sublimate  (in  the  absence  of  neutral  salts)  > 
absolute  alcohol,  and  tannic  acid,  but  the  precipitate  may  redissolve  on  the 
addition  of  an  excess  of  the  precipitant.  As  an  important  difference  between 
amphopeptone  and  antipeptone  we  must  also  mention  that  the  first  gives 
Millon's  reaction,  while  the  antipeptone  does  not. 

In  regard  to  the  precipitation  by  alcohol  we  must  call  attention  to  the  observa- 
tions of  Frankel  that  not  only  are  the  acid  combinations  of  peptone  (Paal) 
soluble  in  alcohol,  but  also  the  free  peptone,  and  Frankel  has  even  suggested  a 

1  Zeitschr.  f.  Biologie,  26.  See  also  Chittenden  and  Meara,  Journ.  of  Physiol.* 
15,  and  Salkowski,  Zeitschr.  f.  Biologie,  34  and  37. 

2  Xeumeister,  Zeitschr.  f.  Biologie,  24,  26. 

3  Zeitschr.  f.  physiol.  Chem.,  24. 


PROTEOSES  AND  PEPTONES.  41 

method  of  preparation  based  on  this  behavior.     Sen rotter1  has  also  prepared 
crystalline  proteoses  which  were  soluble  in  hot  alcohol,  especially  methyl  alcohol. 

The  views  on  the  hydrolytic  cleavage  products  of  peptic  and  tryptic 
digestion  which  were  accepted  until  a  few  years  ago  have  recently  been 
considerably  modified  in  several  points.  As  this  question  of  peptones 
is  at  the  present  time  undergoing  active  development,  and  as  it  Is  also 
very  complicated  and  still  not  clear  in  many  points,  it  is  at  present 
not  possible  to  give  a  clear,  short,  and  still  comprehensive  discussion 
of  the  subject.     We  can  give  here  only  the  most  important  results. 

The  older  view  that  in  peptic  digestion  only  proteoses  and  peptones  but 
no  simpler  cleavage  products  are  formed  has  been  shown  not  to  be  true. 
The  works  of  Zuxtz,  Pfauxdler,  Salaskix,  Lawroav,  Laxgsteix,2  and 
others  have  shown  that  simpler  products  can  be  produced,  some  whose 
nature  is  still  unknown,  while  others  are  known,  such  as  alanin,  leucin, 
leucinimide,  aminovalerianic  acid,  aspartic  and  glutamic  acids,  phenyl- 
alanin,  tyrosin,  pyrrolidin  carbonic  acid  and  lysin,  and  on  further  cleavage 
indeed  also  oxyphenylethylamine,  tetra-  and  pentamethylendiamine.  It 
has  not  been  possible  to  cause  a  disappearance  of  the  biuret  reaction,  and 
the  appearance  of  tryptophan  3  has  only  been  observed  on  using  certain 
apparently  impure  pepsin  preparations.  Pepsin  digestion  therefore  yields 
to  all  appearances  the  same  products  as  obtained  on  hydrolysis  with  min- 
eral acids. 

In  connection  with  the  experimental  results  it  must  be  remarked  that  in 
certain  cases  impure  pepsin  was  used,  or  indeed  autodigestion  of  the  stomach 
mucosa  was  carried  on,  and  that  consequently  the  action  of  pseudopepsin  (see 
Chapter  IX)  was  not  excluded.  In  other  cases  the  digestion  with  pepsin  and 
considerable  acid  (even  1  per  cent  H,S04)  was  continued  for  a  very  long  time, 
indeed  for  an  entire  year,  without  controlling  the  influence  of  the  acid  alone  upon 
the  proteoses. 

Kuhxe's  view  that  in  tryptic  digestion  always  a  peptone,  so  called 
antipeptone,  remains  which  cannot  be  further  split  is  not  strictly  true. 
By  sufficiently  long  autodigestion  of  the  pancreas  Kutscher  4  was  able  to 
obtain  as  final  products  a  mixture  of  digestion  products  which  failed  to 
respond  to  the  biuret  test.  The  pure  antipeptone  (see  below),  isolated 
by  Siegfried,  can  only  be  split  by  trypsin  with  difficulty.  We  do  not 
know  howT  the  antipeptone  prepared  according  to  Kuhxe  from  antialbumid 

1  Frankel,  Zur  Kenntnisse  der  Zerfallsprodukte  des  Eiweisses  bei  peptischer  und 
tryptischer  Yerdauung.     "Wien,  1S9G; — Schrotter,  Monatshefte  f.  Chem.,  11,  16. 

2  Zuntz,  Zeitschr.  f.  physiol.  Chem.,  2S,  and  Hofmeister's  Beitriiire,  2:  Pfaundler, 
Zeitschr.  f.  physiol.  Chem.,  30;  Salaskin,  ibid.,  32#;  Salaskin  and  Kowalewsky,  ibid., 
3S;  Lawrow,  ibid.,  33;  Langstein,  Hofmeister's  Beitrage,  1  and  2. 

3  See  Malfatti,  Zeitschr.  f.  physiol.  Chem.,  31. 

4  Zeitschr.  f.  physiol.  Chem.,  25,  2G,  28,  and  "Die  Endprodukte  der  Trypsinver- 
dauung,"  Ilabilitationsschrift,  Strassburg,  1S99. 


42  THE  PROTEIN  SUBSTANCES. 

acts  iia  this  regard.  The  complete  disappearance  of  the  biuret  reaction 
in  tiyptic  digestion  does  not  show  that  a  complete  destruction  into  simple 
carbon  nuclei  has  taken  place.  According  to  FJ.  Fischer  and  Abder- 
halden,1  polypeptide-like  bodies  are  produced,  especially  in  tryptic  diges- 
tion, and  these  bodies  resist  the  prolonged  action  of  the  enzyme,  but  yield 
several  different  amino  acids  on  hydrolytic  cleavage  by  acids.  The  same 
is  probably  also  true  for  peptic  digestion  (see  below),  and  the  difference 
in  the  digestive  products  between  pepsin  and  trypsin  digestion  consists 
essentially  only  in  that  in  the  first  case  the  cleavage  is  slower  and  does  not 
proceed  so  far  because  the  biuret  reaction  remains  and  no  formation  of 
tryptophan  takes  place. 

By  the  use  of  the  methods,  as  specially  worked  out  by  the  Hofmeister 
school,  of  fractionally  salting  out  with  ammonium  sulphate  or  zinc  sulphate, 
numerous  experiments  have  recently  been  made  to  separate  the  various 
proteoses  and  peptones  by  Umber,  Alexander,  Pfaundler,  and  especially 
by  Pick  and  Zunz.2  Not  only  have  we  learned  by  these  methods  of  a  larger 
number  of  proteoses,  but  our  older  conception  of  the  primarily  produced 
products  has  been  materially  modified.  Immediately  at  the  commence- 
ment of  digestion,  also  in  peptic  digestion,  a  splitting  of  the  proteid  mole- 
cule into  several  complexes  takes  place.  In  opposition  to  the  view  of 
Huppert  3  that  the  proteoses,  in  pepsin  digestion,  are  always  derived 
from  the  primarily  formed  acid  albuminate,  Pick  and  Zunz  have  shown 
that  acid  albuminate  as  well  as  several  proteoses  occurs  primarily  in  the 
commencement  of  the  digestion.  According  to  Goldschmidt  4  a  splitting 
off  of  proteoses  and  the  formation  of  acid  albuminate  takes  place  simul- 
taneously by  the  action  of  dilute  acids  alone.  Besides  the  proteoses  we 
have,  according  to  Zunz  and  Pfaundler,  even  at  the  beginning,  also 
other  primary  bodies,  which  cannot  be  salted  out  and  which  do  not  give 
the  biuret  reaction,  but  are  in  part  precipitated  by  phosphotungstic  acid. 
These,  not  closely  studied,  products  seem  to  be  intermediate  between 
the  peptones  and  the  amino  acids,  and  they  correspond  probably  to  the 
polypeptide-like  bodies  obtained  by  Fischer  and  Abderhalden  in  tryptic 
digestion. 

By  fractional  precipitation  of  Witte's  peptone  with  ammonium  sulphate 
Pick  has  obtained  various  chief  fractions  of  proteoses.  The  first  contains  the 
proto-  and  heteroproteoses,  whose  precipitation  limit  lies  at  24-42  per  cent  satu- 

1  Zeitschr.  f.  physiol.  Chem.,  39. 

2  Umber,  Zeitschr.  f.  physiol.  Chem.,  25;  Alexander,  ibid.,  25;  Pfaundler,  ibid., 
30;  Zunz,  ibid.,  28,  and  Hofmeister 's  Beitriige,  2;  Pick,  ibid.,  2,  and  Zeitschr.  f.  physiol. 
Chem.,  24  and  28. 

3  Schiitz  and  Huppert,  Pfluger's  Arch.,  80. 

4  P.  Goldschmidt,  Ueber  die  einwirkung  von  Sauren  auf  Eiweissstoffe,  Inaug.- 
Diss.  Strassburg,  1898. 


PROTEOSES  AND  PEPTONES.  43 

ration  with  ammonium-sulphate  solution,  i.e.,  the  presence  of  24—42  c.  c.  ammo- 
nium-sulphate solution  in  KM)  c  c  of  the  liquid.  Then  follows  a  fraction  A  at 
54-62  per  cent  saturation,  then  a  third  fraction  B,  with  70-95  per  cent  satura- 
tion, and  finally  fraction  C,  which  precipitates  from  the  saturated  solution  on 
acidification  with  sulphuric  acid  saturated  with  the  salt. 

The  hetercr-  ami  protoproteoses  are  not,  according  to  our  present  views, 
the  only  primary  proteoses.  In  the  proteose  fraction  B  obtained  on  saturat- 
ing with  ammonium  sulphate  in  neutral  liquids,  primary  proteoses  are 
also  found.  To  mention  examples:  the  glucoproteose  (Pick)  which 
contains  a  carbohydrate  group  and  Hofmeister's  *  synproteose.  An 
unequal  power  of  being  salted  out  is  no  longer  sufficient  to  differentiate 
between  the  primary  and  secondary  proteoses. 

We  cannot  enter  into  a  discussion  of  the  various  proteoses  or  proteose- 
fractions.  The  differences  which  exist  between  the  hetero-  and  proto- 
proteoses obtained  from  fibrin  (Pick)  are  of  great  interest.  The  hetero- 
proteose  is  insoluble  in  32  per  cent  alcohol,  yields  only  very  little  tyrosin 
or  indol,  but  gives  abundant  leucin  and  glycocol,  and  contains  about  39 
per  cent  of  the  total  nitrogen  in  a  basic  form.  The  protoproteose,  on 
the  contrary,  is  soluble  in  80  per  cent  alcohol,  yields  considerable  tyrosin 
and  indol,  only  little  leucin,  but  no  glycocol,  and  contains  only  about  25 
per  cent  basic  nitrogen.  Friedmann  and  Hart  2  have  obtained  very  similar 
results  in  regard  to  the  basic  nitrogen  in  the  two  proteoses.  Hart  also 
showed  that  the  heteroproteose  (from  muscle  syntonin)  is  considerably 
richer  in  arginin  and  poorer  in  histidin  than  the  protoproteose. 

According  to  Pick,  the  heteroproteose  is  also  more  resistant  towards 
trypsin  digestion  than  the  protoproteose,  a  behavior  which  coincides  with 
Kdhne  's  view  of  a  resistant  atomic  complex,  an  antigroup  in  the  proteid 
bodies.  Kuhne  and  Chittenden3  obtained  regularly  on  the  tryptic  di- 
gestion of  heteroproteose  a  separation  of  so-called  antialbumid,  a  body 
which  is  attacked  with  great  difficulty  in  tryptic  digestion,  but  which  sepa- 
rates as  a  jelly-like  mass  and  which  is  richer  in  carbon  (57.5-5S.09  per 
cent),  but  poorer  in  nitrogen  (12.61-13.94  per  cent),  than  the  original  pro- 
teid. 

This  antialbumid  has  recently  attracted  further  attention,  because  as 
first  found  by  Danilewski  and  other  investigators,  Okuxkw,  Sawjalow, 
Lawrow,  and  Salaskin  and  Kurajeff  have  further  shown  that  solutions  of 
rennin,  gastric  juice,  pancreatic  juice,  and  papain  solutions  cause  a  similar 
coagulum  in  not  too  dilute  proteose  solutions.  These  eoagula,  called  plas- 
teincs  (coagulum  by  rennin)  by  Sawjalow,  and  coagidoscs  (coagulum  by 


1  Uebcr  Bau  und  Gruppirung  der  Ehveisskorper,  Ergebnisse  der  Phvsiol.,  1,  Abt 
1,783. 

2  Friedmann,  Zeitschr.  f.  physiol.  Chem.,  29;  Hart,  ibid.,  33. 
8  Kuhne  and  Chittenden,  Zeitschr  f.  Biologic,  19,  20. 


44  THE  PROTEIN  SUBSTANCES. 

papain)  by  Kurajeff,1  are  similar  in  many  respects  to  antialbumid,  having  a 
higher  content  of  carbon  (57-60  per  cent)  and  nitrogen  (13-14.6  per  cent). 
They  are  only  produced  from  proteoses  and  not  from  peptones,  and  only  form 
a  small  fraction  of  the  related  proteose.  We  cannot  state  anj^thing  with 
positiveness  for  the  present  in  regard  to  their  importance.  It  is  evident 
from  their  composition  that  they  do  not  represent  the  reformation  of  proteid 
from  the  proteoses,  as  claimed  by  some  investigators. 

The  method  of  fractional  precipitation  by  ammonium  or  zinc  sulphate 
has  undoubtedly  been  of  the  greatest  service  in  the  study  of  the  diges- 
tive products.  Still,  the  practice  of  calling  those  products  which  can 
be  salted  out,  proteoses,  and  those  which  cannot  be  salted  out,  peptones, 
has  led  to  many  mistakes  and  to  a  complete  misunderstanding  of  the 
peptone  question.  Originally  we  considered  those  bodies  peptones  which 
still  had  a  positive  proteid  nature/  but  now  we  indicate  as  peptones  all 
digestive  products  which  cannot  be  salted  out,  but  which  still  give  the  biuret 
reaction  (although  the  biuret  reaction  does  not  of  necessity  show  the  pro- 
teid nature  of  a  substance).  J  The  nature  of  these  peptones  is  still  unknown. 

It  is  also  generally  admitted  that  the  peptones  are  mostly  mixtures 
of  various  bodies.2  Only  those  peptones  isolated  by  Siegfried  and  his 
pupils  Muhle,  Fr.  Muller,  Borkel,  and  Kruger3  must  be  considered 
as  chemical  individuals.  All  these  peptones  are  acids  which  form  salts 
with  carbonates  with  the  evolution  of  carbon  dioxide;  they  are  lsevorotatory 
and  have  a  constant  rotation.  The  pepsin-fibrin  peptones  a  and  /?  isolated 
and  studied  by  Siegfried,  Muhle,  and  Borkel  have  the  following  formulae, 
C21H31N609  and  C21H36N6O10,  respectively.  The  ^-peptone  seems  to  be 
converted  into  a-peptone  on  splitting  off  of  water.  These  pepsin-peptones 
give  the  biuret  test  as  well  as  Millon's  reaction.  Their  solutions  are  not 
precipitated  by  tannic  acid,  picric  acid,  corrosive  sublimate,  phospho- 
tungstic  acid,  and  alcohol,  but  are  precipitated  by  basic  lead  acetate,  meta- 
phosphoric  acid,  or  acetic  acid  and  potassium  ferrocyanide.  The  pepsin- 
peptone  may  be  considered  as  an  amphopeptone  in  Kuhne's  sense,  as  in 
trypsin  digestion  amino  acids  are  formed  and  all  the  tyrosin  and  arginin 
is  split  off  and  antipeptone  is  formed. 

The  trypsin-fibrin  peptones  studied  by  Siegfried  and  Muller  have  the 
formulas  a,  CI0H17N3O5  and  /?,  CuH10N3O5.     They  have  a  different  specific 

1  The  works  of  Danielewski  and  Okunew  are  cited  and  reviewed  in  the  following : 
Sawjalow,  Pfliiger's  Arch.,  85,  and  Centralbl.  f.  Physiol.,  16;  Lawrow  and  Salaskin, 
Zeitschr.  f.  physiol.  Chem.,  36;  Kurajeff,  Hof meister 's  Beitriige,  1  and  2;  see  also 
Sacharow,  Biochem.  Centralbl.,  1,233;  see  also  Bayer,  Hofmeister's  Beitriige,  4,  in 
regard  to  plastein. 

2  See  Kutcher,  1.  c. ;  Friinkel  and  Langstein,  Wien.  Sitzungsber.  Math.-Naturw, 
Klasse,  110,  1901;   Pick,  Hofmeister's  Beitriige,  2. 

'Siegfried,  Arch.  f.  (Anat.  u.)  Physiol.,  1894;  also  Zeitschr.  f.  physiol.  Chem.,  21g 
Siegfried  and  his  pupils,  ibid.,  38. 


PEPTONES.  45 

rotation.  The  fad  that  two  different  antipeptones  are  formed  from  the 
pepsin-fibrin  peptone  shows  that  this  latter  contains  at  least  two  anti- 
groups,  and  not,  as  Kuhne  claimed,  only  one.  The  antipeptones  do  not 
give  the  biuret  test,  but  respond  to  Mi  LLON's  reaction,  and  contain  notyrosin 
groups.  They  are  precipitated  by  alcohol  and  are  precipitated  less  readily 
or  less  completely  by  the  reagents  which  precipitate  the  pepsin-peptones. 
They  have  a  persistent  resistance  towards  further  cleavage  by  trypsin. 
On  hydrolysis  with  mineral  acids  they  yield  arginin,  lysin,  glutamic  acid, 
and  it  seems  also  aspartic  acid.  The  quantity  of  basic  nitrogen  is  less 
than  25  per  cent  and  the  nitrogen  split  off  as  ammonia  in  antipeptone 
is  p  16.1  and  a  21.9  per  cent  of  the  total  nitrogen. 

The  glutin-peptones  isolated  by  Siegfried  and  Kruger  have  the 
formula?  C,3H39X7O10  for  the  pepsin-glutin  peptone  and  (^H^NgO,  for 
the  j'-trvpsin-glutin  peptone.  From  the  latter  Siegfried,  on  warming 
with  hydrochloric  acid,  obtained  a  base,  C^H^NgOg,  which  can  also  be 
directly  obtained  from  gelatine,  which  is  called  a  kyrin  because  it  is  to 
be  considered  as  a  basic  protein  nucleus,  and  which  he  therefore  calls 
glutok'jrin.  The  glutokyrin  gives  the  biuret  reaction  and  is  considered 
as  a  basic  peptone.  On  complete  hydrolytic  cleavage  it  yields  arginin, 
lysin,  glutamic  acid,  and  a  second  amino  acid  (probably  glycocoll).  Of 
the  total  nitrogen  two  thirds  belongs  to  the  bases  and  one  third  to  the 
amino  acids.  On  hydrolysis  of  silk  fibroin  successively  with  hydrochloric 
a<id.  trypsin,  and  barium  hydrate,  E.  Fischer  *  also  obtained  peptone- 
like substances  and  finally  a  dipeptide-like  body,  probably  glycylalanin. 

On  account  of  the  cleavage  taking  place  in  digestion  the  digestive 
products  must  have  a  lower  molecular  weight  than  the  original  proteid. 
This  is  also  the  case.  The  molecular  weight  of  the  different  proteids  has 
not  been  determined  with  certainty,2  but  it  is  generally  considered  as  about 
5000-7000  for  the  albumins  and  for  casein.  The  molecular  wreight  for  proto- 
proteoses  was  found  by  Sabanejew  to  be  2467-2643  and  3200  for  the 
deuteroproteoses.  The  peptones  have  a  still  lower  molecular  weight,  being 
between  400  and  250  for  the  various  peptones  (Sabanejew,  Paal,  Sjoqvist3). 

The  elementary  analyses  *  have  not  given  us  much  information  as  to 
the  characteristic  differences  between  the  various  proteoses  and  most 
so-called  peptones.     Certain  proteoses,  especially  those  that  can  be  salted 

1  Siegfried,  Kgl.  Sachs.  Ges.  d.  Wiss.  Math.  Phys.  Klasse,  1903;  Pischer,  see  Bio- 
chem.  Centralbl.,  1,  84. 

:  See  especially  F.  N.  Schulz,  Die  Grosse  die  Eiweissmolekiils,  Jena,  1903. 

3  Sabanejew,  Ber.  d.  d.  chem.  Gesellsch.,  26,  385;  Paal,  ibid.,  27,  1S27;  Sjoqvist, 
Skand.  Arch.  f.  Physiol.,  5. 

4  Elementary  analyses  of  proteoses  and  peptones  will  be  found  in  the  works  of 
Kuhne  and  Chittenden  and  their  pupils,  cited  in  foot-note  3,  page  39;  also  by  Herth, 
Zeitschr.  f.  physiol.  Chem.,  1,  and  Monatshefte  f.  Chem.,  o;  Maly,  Pfliiger's  Arch., 
9.  20;   Henninger,  Compt.  rend.,  S6;   Schrotter,  1.  c.;   Paal,  1.  c. 


46  THE  PROTEIN  SUBSTANCES. 

out  with  difficulty,  and  the  peptones  differ  very  materially  in  composition 

from  the  mother  substances  and  often  have  a  lower  carbon  content. 

Besides  the  behavior  of  being  salted  out  attempts  have  been  made  to 
find  other  points  of  difference  between  the  peptones  and  proteoses.  Schrotter 
and  Frankel1  consider  the  sulphur  content  as  a  pronounced  point  of  difference. 
The  peptones,  according  to  them,  are  free  from  sulphur,  while  the  proteoses,  on 
the  contrary,  contain  sulphur.  Frankel  has  only  been  able  to  find  one  proteose 
(in  Kuhne's  sense)  which  did  not  contain  sulphur. 

In  the  preparation  and  separation  of  various  proteoses  and  peptones  all 
precipitable  proteid  is  always  first  removed  by  neutralization  and  then  by 
boiling.  The  proteoses  may  then  be  separated  from  the  peptones  by  means 
of  ammonium  sulphate  according  to  Kuhne's  method  and  divided  into 
different  fractions  according  to  Pick  and  the  Hofmeister  school.  The 
separation  and  preparation  of  pure  hetero-  and  protoproteoses  can  be 
best  performed  by  the  method  suggested  by  Pick.2  As  in  the  preparation 
of  different  proteoses  and  peptones  we  are  not  dealing  in  most  cases  with 
pure  substances,  but  with  mixtures  or  fractions  it  is  perhaps  sufficient  to 
simply  call  attention  here  to  other  methods  such  as  those  suggested  by  K. 
Baumann  and  Bomer,  P.  Muller,  Frankel,  Schrotter,  and  Paal.  The 
only  method  which  seems  thus  far  to  have  led  to  a  pure  preparation  of 
peptone  seems  to  be  the  iron  method  used  by  Siegfried.3 

For  the  detection  of  proteoses  and  peptones  in  animal  fluids  we  proceed 
as  follows,  according  to  Devoto:  The  coagulable  proteids  are  removed  by 
prolonged  heating,  and  the  solution  is  then  saturated  with  ammonium  sul- 
phate. True  peptones  (besides  deuteroproteose  not  precipitated)  may  be 
detected  in  the  cold  filtrate  by  means  of  the  biuret  test.  The  proteoses 
are  contained  in  the  mixture  of  precipitate  and  salt  crystals  collected  on 
the  filter.  The  proteoses  are  dissolved  from  this  mixture  by  washing  with 
water,  and  may  be  detected  in  the  wash-water  by  means  of  the  biuret 
test.  According  to  Halliburton  and  Colls  4  traces  of  proteoses  may 
be  formed  from  other  proteids  in  this  method  by  prolonged  heating. 
As  the  best  methods  they  suggest  either  the  precipitation  of  the  native 
proteids  by  the  addition  of  a  10  per  cent  trichloracetic  acid  solution  or  by 
making  the  native  proteids  insoluble  by  the  continuous  action  of  alcohoL 
The  last  method  is  not  quite  applicable  to  blood-serum,  as  the  so-called 
fibrin-ferment,  which  also  gives  the  biuret  test,  is  not  made  insoluble  by 
this  procedure. 

If  a  solution  saturated  with  ammonium  sulphate  is  to  be  tested  by  the 
biuret  test,  it  must  first  be  treated  with  a  slight  excess  of  concentrated 
caustic-soda  solution,  keeping  the  solution  cold,  and  after  the  sodium 
sulphate  has  settled  the  liquid  is  treated  with  a  2  per  cent  solution  of 
copper  sulphate,  drop  by  drop. 

The  biuret  test  (colorimetric)  and  the  polariscopic  method  have  been 

1  Schrotter,  Monatshcfte  f.  Chem.,  14  and  10;  Frankel,  Zur  Kenntnis  der  Zerfalls- 
produkte  des  EiweLss  bei  peptiflcher  und  tryptischer  Verdauung,  Wien,  1896. 
:'  Kiihnc,  Zeitschr.  f.  Biologie,  28;   Pick,  1.  c. 

3  Baumann  and  Burner,  Chem.  Centralbl.,  1898,  1,  040;  Muller,  Zeitschr.  f.  physiol. 
Chem.,  20;  Friinkel,  1.  c,  Zur  Kenntnis,  etc.;  Schrotter,  Monatshefte  f.  Chem.,  14 
and  10;   Paal,  1.  c. ;  Siegfried,  1.  c. 

4  Devoto,  Zeitschr.  f.  physiol.  Chem.,  15;  Halliburton  and  Colls,  Journ.  of  Path, 
and  Bact.,  1895. 


PROTAMINS.  47 

used  in  the  quantitative  estimation  of  proteoses  and  peptones.     These 
methods  do  not  yield  exact  results. 

Coagulated  Proteids.  [Proteids  may  be  converted  into  the  coagulated 
condition  by  different  means:  by  heating,  by  the  action  of  alcohol,  especially 
in  the  presence  pf  neutral  salts,  by  chloroform,  ether,  metallic  salts,  and  by 
the  prolonged  shaking  of  their  solutions  (Ramsden  *),  and  in  certain  cases, 
as  in  the  conversion  of  fibrinogen  into  fibrin  (Chapter  VI),  by  the  action  of 
an  enzyme.  The  nature  of  the  processes  which  take  place  during  coagula- 
tion is  unknown.  The  coagulated  albuminous  bodies  are  insoluble  in 
water,  in  neutral  salt  solutions,  and  in  dilate  acids  or  alkalies,  at  normal 
temperature.  They  are  dissolved  and  converted  into  albuminates  by 
the  action  of  less  dilute  acids  or  alkalies,  especially  on  heating^) 

Coagulated  proteids  also  seem  to  occur  in  animal  tissues.  We  find,  at 
least  in  many  organs  such  as  the  liver  and  other  glands,  proteids  which  are 
not  soluble  in  water,  dilute  salt  solutions,  or  very  dilute  alkalies,  and  only 
dissolve  after  being  modified  by  strong  alkalies. 

Appendix. 
PROTAMINS   AND   HISTONS. 

I.  Protamins.  In  close  relationship  to  the  proteids  stands  a  group  of 
substances,  the  protamins;  discovered  by  Miescher,  which  are  designated" 
by  Kossel  as  the  simplest  proteids  or  as  the  nucleus  of  the  protein  bodies. 
Thus  far  they  have  only  been  found  in  combination  with  nucleic  acids 
in  fish  spermatozoa.  They  differ  essentially  from  the  proteids  by  the 
fact  that  they  yield  chiefly  diamino  acids  (always  abundant  arginin)  as 
cleavage  products  and  only  very  little  monamino  acids.  They  are  strong 
basic  substances  rich  in  nitrogen  (about  30  per  cent  or  more)  and  have 
high  molecular  weight. 

Protamin  was  discovered  by  Miescher  2  in  salmon  spermatozoa.  Later 
Kossel  isolated  and  studied  similar  bases  from  the  spermatozoa  of  herring, 
sturgeon,  mackerel,  and  other  fishes.  As  all  these  bases  are  not  identical, 
Kossel  uses  the  name  protamins  to  designate  the  group  and  calls  the 
individual  protamins  according  to  their  origin  salmin,  clupcin,  scombrin, 
sturin,  accipenserin,  cyclopterin,  etc. 

The  percentage  composition  of  these  bodies  has  not  been  satisfactorily 
determined.      As     probable    formulae    we    have    for     salmin    C32H5BN,804 

1  Du  Bois-Reymond's  Arch.,  1894. 

2  In  regard  to  protamins,  see  Miescher  in  the  histo-chemical  and  physiological  works 
of  Fr.  Miescher,  Leipzig,  1S97;  Piccard,  Ber.  d.  deutsch.  chem.  Gesellsch..  7  ;  Schmiede- 
berg,  Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Kossel,  Zeitschr.  f.  physiol.  Chem.,  22  (Ueber 
die  basischen  Stoffe  des  Zellkerns)  and  25,  165  and  190,  and  Sitzungsber.  der  Gesellsch. 
zur  Beford.  der  ges.  Naturwiss.  zu  Marburg,  1S97;  Kossel  and  Mathews,  Zeitschr.  f. 
physiol.  Chem.,  23  and  25;  Kossel  and  Kutscher,  ibid., SI;  Goto,  ibid.,  37;  Kurajeff, 
ibid.,  32;   Morkowin,  ibid.,  2$. 


48  THE  PROTEIN  SUBSTANCES. 

(Miescher-Schmiedeberg)  or  C^H^N^O,,  (Kossel  and  Goto),  for  clupein 
C30H62N14O9,  and  for  sturin  C36H6tfN1907  (Kossel)  or  C34H7,N1709  (Goto). 
On  boiling  with  dilute  mineral  acids  as  also  by  tryptic  digestion  the 
protamins  first  yield  peptone-like  substances  called  protones,  from  which 
simpler  products  are  derived  on  further  cleavage.  Salmin  and  clupein 
yield  arginin  (84.3  or  82.2  per  cent),  aminovalerianic  acid,  and  an  unknown 
residue.  Cyclopterin  yields  about  62.5  per  cent  arginin,  about  8  per  cent 
tyrosin,  besides  other  unknown  bodies.  Sturin  on  the  contrary  yields  all 
three  hexon  bases,  12.9  per  cent  hisidin,  58.2  per  cent  arginin,  and  12  per 
■cent  lysin,  and  besides  these  an  unknown  monamino  acid.  It  contains  for 
•even-  molecule  of  histidin  one  molecule  of  lysin  and  four  molecules  of  arginin. 

The  more  recent  investigations  of  Kossel  and  Kossel  and  Dakin  *  on 
the  protamins  have  shown  that  salmin  yields  arginin,  a-pyrrolidin  car- 
bonic acid,  aminovalerianic  acid  and  serin  as  cleavage  products.  Clupein 
yields  arginin,  aminovalerianic  acid,  serin,  and  probably  also  a-pyrrolidin 
carbonic  acid.  The  spermatozoa  of  the  carp  contain  two  different  pro- 
tamins, a- and  /?-cyprinin.  The  a-cyprinin  contains  little  arginin  (4.9  per 
cent)  and  considerable  lysin  (28.8  per  cent).  The  /?-cyprinin  is  like  the 
other  protamins,  rich  in  arginin  and  poorer  in  lysin.  It  also  contains 
tyrosin,  which  only  exists  in  the  a-cyprinin  to  a  very  trivial  extent,  and 
probably  originates  in  this  case  from  a  contamination  with  /?-cyprinin. 

Solutions  of  these  bases  in  water  are  alkaline  and  have  the  property 
of  giving  precipitates  with  ammoniacal  solutions  of  proteids  or  primary  pro- 
teoses. These  precipitates  are  considered  as  histons  by  Kossel.  The  salts 
with  mineral  acids  are  soluble  in  water,  but  insoluble  in  alcohol  and  ether. 
They  are  more  or  less  readily  precipitated  by  neutral  salts  (NaCl).  Among 
the  salts  of  the  protamins  the  sulphate,  picrate,  and  the  double-platinum 
chloride  are  the  most  important  and  are  used  in  the  preparation  of  the 
protamins.  The  protamins  are  like  the  proteids,  lsevogyrate.  They  give 
the  biuret  test  beautifully,  but  with  the  exception  of  c}^clopterin  do  not 
give  Millox's  reaction.  The  protamin  salts  are  precipitated  in  neutral 
or  even  faintly  alkaline  solutions  by  phosphotungstic  acid,  tungstic  acid, 
picric  acid,  chromic  acid,  and  alkali  ferrocyanides. 

The  protamins  are  prepared,  according  to  Kossel,  by  extracting  the 
heads  of  the  spermatozoa,  which  have  previously  been  extracted  with 
alcohol  and  ether,  with  dilute  sulphuric  acid  (1-2  per  cent),  filtering,  and 
precipitating  with  4  vols,  of  alcohol.  The  sulphate  may  be  purified  by 
repeated  solution  in  water  and  precipitation  with  alcohol,  and  if  necessary 
conversion  into  the  picrate.  For  more  details  see  the  works  of  Kossel. 
The  double-platinum  salt  is  best  suited  for  analysis  and  can  be  obtained 
according  to  Goto  by  precipitating  the  methyl-alcohol  solution  of  the 
protamin  hydrochloride  with  platinum  chloride.  Miescher  also  precipitates 
the  base  as  a  double  platinum  salt. 

1  Zeitschr.  f.  physiol.  Chcm.,  40;  Kossel  and  Dakin,  ibid. ,  40. 


HISTONS.  49 

2.  Histons  are  also  basic  protcids  which  stand  to  a  certain  extent 
between  the  protamins  and  the  true  proteids,  Their  content  of  nitrogen 
varies  between  16.5  and  19.8  per  cent  and  in  certain  instances  is  not  higher 
than  in  other  proteids,  especially  vegetable  protcids.  According  to  Kossel 
and  KuTSCHEB  and  Lawrow  they  are  on  the  contrary  richer  in  basic  nitrogen 
ami  especially  yield  more  arginin  than  other  proteids.  Kossel  first  isolated 
a  peculiar  protein  substance  from  the  red  corpuscles  of  goose  blood  which 
was  precipitated  by  ammonia,  and  because  of  its  similarity  in  certain  regards 
to  the  peptones  (in  the  old  sense)  he  called  it  histon.  At  the  present  time 
a  number  of  very  different  bodies  are  described  as  histons,  such  as  those 
obtained  from  nucleohiston  (Liliexfeld),  from  haemoglobin  (globin  accord- 
ing to  ScHULz),from  mackerel  spermatozoa  (scombron  according  to  Banc), 
from  the  codfish  (gadushiston  according  to  Kossel  and  Kutscher),  from 
the  frogs,  etc.  (lotahiston,  Ehrstrom),  and  from  the  sea-urchin  (arbacin, 
Mathews).1 

Sulphur  has  been  found  in  those  histons  in  which  it  has  been  tested  for. 
They  give  the  biuret  test,  but  as  a  rule  only  a  faint  Millon  's  reaction.  The 
goose-blood  histon  first  studied  by  Kossel  gives  the  following  three  reac- 
tions: The  neutral  salt-free  solution  first,  does  not  coagulate  on  boiling, 
second,  gives  a  precipitate  with  ammonia  which  is  insoluble  in  an  excess 
of  the  precipitant,  third,  gives  a  precipitate  with  nitric  acid  which  disap- 
pears on  heating  and  reappears  on  cooling. 

The  different  histons  behave  differently  towards  these  three  reactions, 
and  hence  they  are  not  specific.  On  the  other  hand,  all  histons  seem  to 
be  precipitated  from  neutral  solution  by  alkaloid  reagents,  and  they  also 
produce  precipitates  in  proteid  solutions.  These  two  reactions  are  also 
not  specific  for  the  histons,  as  the  protamins  have  a  similar  behavior.  The 
histons  differ  from  the  protamins  by  a  much  lower  content  of  basic  nitrogen, 
and  also  probably  by  always  containing  sulphur.  True  proteids,  as  Os- 
borxe's2  edestan,  also  give  these  two  reactions;  therefore  it  is  impossible 
by  qualitative  reactions  alone  to  identify  a  substance  as  a  histon  with 
positiveness.  The  large  content  of  basic  nitrogen  and  arginin  is  not  a 
sure  point  of  difference  between  histons  and  other  bodies.  Histon  yields 
little  more  than  40  per  cent  basic  nitrogen,  while  a  heteroproteose  yields 
about  the  same,  namely,  39  per  cent,  Histon  yields  14-15.5  per  cent 
arginin  (gadushiston),  and  the  lotahiston  only  12  per  cent.  The  plant- 
globulin  edestin  3  yields  a  much  larger  amount  of  arginin,  namely,  14.07 

1  Kossel,  Zeitschr.  f.  physiol.  Chem.,  S,  and  Sitzungsber.  der  Gesellsch.  zur  Beford. 
d.  ges.  Wissensch.  zu  Marburg,  1S97;  Kossel  and  Kutscher,  ibid.,  1900,  and  Zeitschr. 
f.  physiol.  Chem.,  31;  Lawrow,  ibid.,  28,  and  Ber.  d.  d.  chem.  Gesellsch.,  34;  Lilienfeld, 
Zeitschr.  f.  physiol.  Chem.,  18;  Schulz,  ibid.,  21;  Bang,  ibid.,  27;  Ehrstrom,  ibid., 
32;   Mathews,  ibid.,  28. 

1  Zeitschr.  f.  physiol.  Chem.,  33. 

8  See  Kossel,  Ber.  d.  d.  chem.  Gesellsch.,  31,  3236. 


50  THE  PROTEIN  SUBSTANCES. 

per  cent.  Ulpiani  1  has  isolated  a  protein  substance  from  the  sper- 
matozoa of  the  thynnus  vulgaris  (a  fish  similar  to  the  mackerel)  which 
stands  between  the  protamins  and  the  histons.  Its  sulphate  contained 
23.94  per  cent  nitrogen.  It  gave  the  biuret  test  as  well  as  Millon's 
reaction  and  yielded  arginin  on  cleavage  with  sulphuric  acid.  Instead  of 
lysin  and  histidin  other  bases  were  present.  If  the  histons  are  inter- 
mediate bodies  between  the  protamins  and  proteids,  it  is  not  to  be  ex- 
pected that  histons  should  have  perfectly  specific  reactions,  and  for  this 
reason  it  is  hardly  possible  for  the  present  to  give  a  precise  definition  for 
the  histons. 

The  parahiston  found  by  Fleroff  in  the  thymus  gland  yields  so  little  basic 
nitrogen  that  it  probably  does  not  belong  to  the  histon  group   (Kossel  and 

KUTSCHER  2). 

II.  Compound  Proteids. 

With  this  name  we  designate  a  class  of  bodies  which  are  more  complex 
than  the  proteids  and  which  yield  as  nearest  splitting  products  proteids 
on  the  one  side  and  non-proteid  bodies,  such  as  pigments,  carbohydrates, 
nucleic  acids,  etc.,  on  the  other.3 

The  compound  proteids  known  at  the  present  time  are  divided  into 
three  chief  groups.  These  are  the  hcemoglobins,  the  glucoproteids,  and  the 
nucleoproteids.  The  haemoglobins  will  be  treated  in  a  following  chapter 
(Chapter  VI),  on  the  blood. 

Glucoproteids  are  those  compound  proteids  which  on  decomposition 
yield  a  proteid  on  one  side  and  a  carbohydrate  or  derivatives  of  the  same 
on  the  other,  but  no  purin  bodies.  Some  glucoproteids  are  free  from 
phosphorus  (mucin  substances,  chondroproteids,  and  hyalogens),  and 
some  contain  phosphorus  (phosphoglucoproteids). 

The  glucoproteids  free  from  phosphorus  may,  on  account  of  the  carbo- 
hydrate groups  split  off,  be  divided  into  two  chief  groups,  the  mucin  sub- 
stances and  the  chondroproteids.  The  first  yields  in  hydrolytic  cleavage 
an  amino-sugar,  which,  with  one  exception,4  has  been  shown  to  be  glucos- 
amine. In  the  chondroproteids,  on  the  contrary,  the  proteid  is  united 
to  chondroi tin-sulphuric  acid. 

*Gazz.  chim.  Ital.,  32.     Also  Biochem.  Centralblt.,  1. 

2  Fleroff,  Zeitschr.  f.  physiol.  Chem.,  28;  Kosscl  and  Kutscher,  1.  c. 

3  Iloppe-Seyler  has  given  the  name  prote'ide  to  these  compound  proteids,  but  as 
this  term  is  misleading  in  English  we  do  not  use  it  in  English  classifications  in  this 
sense. 

*  See  Schulz  and  Ditthorn,  Zeitschr.  f.  physiol.  Chem.,  29.  When  both  carbo- 
hydrate groups  are  simultaneously  combined  with  one  body,  then  we  are  not  probably 
dealing  with  a  chemical  individual,  but  rather  with  a  mixture. 


MUCINS.  51 

Mucin  Substances.  Those  bodies  contain  carbon,  hydrogen,  nitrogen, 
sulphur,  and  oxygen.  Compared  with  proteids  they  are  poorer  in  nitrogen, 
and  as  a  rule  have  also  considerably  less  carbon.  The  carbohydrate  complex, 
whose  nature  has  been  shown  by  the  investigations  of  Fe.  MiJLLEB  l  and 
his  pupils,  occurs,  as  it  seems,  in  the  mucin  substances,  as  a  polysaccharide, 
related  to  chitosan,  which  on  hydrolytic  cleavage  yields  glucosamine 
(chitosamine),  and,  at  least  in  most  cases,  also  acetic  acid.  The  mucin 
substances  differ  very  markedly  among  one  another,  hence  we  divide  them 
into  two  groups,  the  mucins  and  the  mucoids. 

The  true  mucins  are  characterized  by  their  natural  solution,  or  one 
prepared  by  the  aid  of  a  trace  of  alkali,  being  mucilaginous,  thread-like, 
and  giving  a  precipitate  with  acetic  acid  which  is  insoluble  in  excess  of  acid 
or  soluble  only  with  great  difficulty.  The  mucoids  do  not  show  these 
physical  properties  and  have  other  solubilities  and  precipitation  properties. 
As  we  have  intermediate  steps  between  different  protein  bodies,  so  also 
we  have  such  between  true  mucins  and  mucoids,  and  a  sharp  line  between 
these  two  groups  cannot  be  drawn. 

It  is  just  as  difficult  at  present  to  draw  a  sharp  line  between  the  pro- 
teids and  the  mucins  or  mucoids,  since  we  have  been- able  to  split  off  carbo- 
hydrate complexes  from  several  proteids,  and  as  the  proteids  of  the  white 
of  egg  are  undoubtedly  glucoproteids.  It  is  immaterial  whether  we 
consider  these  glucoproteids  as  belonging  to  the  mucoids  or  to  a  special 
group  or  not.  From  a  comparative  chemical  standpoint  they  undoubtedly 
belong  to  the  mucoid  group,  which  occurs  in  eggs  to  a  considerable  extent. 

True  mucins  are  secreted  by  the  larger  mucous  glands,  by  certain 
mucous  membranes,  also  by  the  skin  of  snails  and  other  animals.  True 
mucin  also  occurs  in  the  navel-cord.  Sometimes,  as  in  snails  and  in  the 
membrane  of  the  frog-egg  (Giacosa  2),  a  mother-substance  of  mucin,  a 
mucinogen,  has  been  found  which  may  be  converted  into  mucin  by  alkalies. 
Mucoid  substances  are  found  in  certain  cysts,  in  the  cornea,  the  crystalline 
lens,  wyhite  of  egg,  and  in  certain  ascitic  fluids.  The1  so-called  tendon 
mucin,  which  according  to  the  recent  investigations  of  Levexe,  Cutter 
and  Gies  3  contains  chondroitin-sulphuric  acid  or  a  related  substance,  cannot 
be  classified  as  a  mucin,  but  must,  like  the  chondromucoid  and  the  osseo- 
mucoid, be  classified  as  chondroproteid.  As  the  mucin  question  has  been 
very  little  studied,  it  is  at  the  present  time  impossible  to  give  any  positive 
statements  in  regard  to  the  occurrence  of  mucins  and  mucoids,  especially 
as  without  doubt  in  many  cases  non-mucinous  substances  have  been  de- 
scribed as  mucins. 

1  See  Fr.  Miiller,  Zeitschr.  f.  Biologie,  42,  which  contains  all  the  relative  literature, 
and  also  L.  Langstein,  Die  Bildung  von  Kohlenhydraten  aus  Eiweiss.  Ergebnisse  der 
Physiologie,  1,  Abt.  1. 

2  Zeitschr.  f.  physiol.  Chem.,  7;   also  Hammarsten,  Pfliiger'a  Archiv,  36. 

1  Levene,  Zeitschr.  f.  physiol.  Chem.,  31;  Cutter  and  Gies,  Amer.  Journ.  of  Physiol.,  6. 


52  THE  PROTEIN  SUBSTANCES. 

i.  True  Mucins.  Thus  far  we  have  been  able  to  obtain  only  a  few 
mucins  in  a  pure  and  unchanged  condition,  due  to  the  reagents  used.  The 
elementary  analyses  of  these  mucins  have  given  the  following  results: 

C          H  N          S 

Mucin  from  mucous  membrane   (air- 
passages) 48.26  6.91  10.7  1.4       (Fr.  Miller) 

Mucin  from  submaxillary 48 .  84  6 .  80  12 .  32  0 .  84     (Hammarsten) 

Mucin  from  snail 50.32  6.84  13.65  1.75     (Hammarsten1) 

Muller  obtained  35  per  cent  glucosamine  from  mucous-membrane 
mucin  and  23.5  per  cent  from  the  submaxillary  mucin. 

By  the  action  of  superheated  steam  on  mucin  a  carbohydrate,  animal 
gum  (Landwehr),  is  split  off.  This  has  not  been  substantiated  by  other 
investigators,  such  as  Folin  and  F.  Muller.2  Instead  of  a  non-nitrogenous 
gum  a  nitrogenous  carbohydrate  derivative  was  always  obtained. 

On  boiling  mucin  with  dilute  mineral  acids,  acid  albuminate  and  bodies 
similar  to  proteoses  are  obtained,  besides  a  reducing  substance  which  is 
not  free  glucosamine  (Steudel).3  By  the  action  of  stronger  acids  we 
obtain  among  other  bodies  leucin,  tyrosin,  and  levulinic  acid.  Certain 
mucins,  as  the  submaxillary  mucin,  are  easily  changed  by  very  dilute 
alkalies,  as  lime-water,  while  others,  such  as  tendon-mucin,  are  not  affected. 
If  a  strong  caustic-alkali  solution,  as  a  5  per  cent  KOH  solution,  is  allowed 
to  act  on  submaxillary  mucin,  we  obtain  alkali  albuminate,  bodies  similar 
to  proteose  and  peptone,  and  one  or  more  substances  of  an  acid  reaction 
and  with  strong  reducing  powers. 

In  one  or  the  other  respect  the  various  mucins  act  somewhat  dissimilarly. 
For  example,  the  snail  and  sputum  mucins  are  insoluble  in  dilute  hydro- 
chloric acid  of  1-2  p.  m.,  while  the  mucin  of  the  submaxillary  gland  and 
the  navel-cord  are  soluble.  The  first  become  flaky  with  acetic  acid, 
while  the  submaxillary  mucin  is  precipitated  in  more  or  less  fibrous,  tough 
masses.     Still  all  the  mucins  have  certain  reactions  in  common. 

In  the  dry  state  mucin  forms  a  white  or  yellowish-gray  powder.  When 
moist  it  forms,  on  the  contrary,  flakes  or  yellowish-white  tough  lumps  or 
masses.  The  mucins  are  acid  in  reaction.  They  give  the  color  reactions  of 
the  proteids.  They  are  not  soluble  in  water,  but  may  give  a  neutral  solu- 
tion with  water  and  the  smallest  quantity  of  alkali.  Such  a  solution  does 
not  coagulate  on  boiling,  while  acetic  acid  gives  at  the  normal  temperature 
a  precipitate  which  is  nearly  insoluble  in  an  excess  of  the  precipitant.  If 
5-10  per  cent  NaCl  be  added  to  a  mucin  solution,  this  can  now  be  carefully 
acidified  with  acetic  acid  without  giving  a  precipitate.     Such  acidified  solu- 

1  Fr.  Muller,  Zeitschr.  f.  Biologie,  42;   Hammarsten,  Pfliiger's  Arch.,  36. 

2  Landwehr,  Zeitschr.  f.  physiol.  Chem.,  8,9;  also  Pfliiger's  Arch.,  39  and  40; 
Folin,  Zeitschr.  f.  physiol.  Chem.,  23;  Fr.  Muller,  Sitzungsber.  d.  Gesellsch.  zur  Beford. 
d.  gesammt.  Natunviss.  zu  Marburg,  1896. 

3  Zeitschr.  f.  physiol.  Chem.,  34. 


MUCOIDS.  53 

tions  are  copiously  precipitated  by  tannic  acid;  with  potassium  forrocyanido 
they  give  no  precipitate,  but  on  sufficient  concentration  they  become  thick 
or  viscous.  A  neutral  solution  of  mucin-alkali  is  precipitated  by  alcohol 
in  the  presence  of  neutral  salts;  it  is  also  precipitated  by  several  metallic 
salts.  If  mucin  is  heated  on  the  water-bath  with  dilute  hydrochloric  acid 
of  about  2  per  cent,  the  liquid  gradually  becomes  a  yellowish  or  dark  brown 
and  reduces  cuprous  oxide  from  alkaline  solutions. 

The  mucin  most  readily  obtained  in  large  quantities  is  the  submaxillary 
mucin,  which  may  be  prepared  in  the  following  way:  The  filtered  watery 
extract  of  the  gland,  free  from  form-elements  and  as  colorless  as  possible, 
is  treated  with  25  per  cent  hydrochloric  acid,  so  that  the  liquid  contains 
1.5  p.  m.  11(1.  On  the  addition  of  the  acid  the  mucin  is  immediately  pre- 
cipitated, but  dissolves  on  stirring.  If  this  acid  liquid  is  immediately 
diluted  with  2-3  vols,  of  water,  the  mucin  separates  and  may  be  purified 
by  redissolving  in  1-5  p.  m.  acid,  and  diluting  with  water  and  washing  there- 
with. The  mucin  of  the  navel-cord  may  be  prepared  in  the  same  way.1  As 
a  rule  the  mucins  can  be  prepared  by  precipitation  with  acetic  acid  and 
repeated  solution  in  dilute  lime-water  or  alkali  and  reprecipitation  with 
acetic  acid.  Finally  they  are  treated  with  alcohol  and  ether.  In  the 
preparation  of  sputum  mucin  a  very  complicated  method  is  necessary 
(Fr.  Mullbr). 

2.  Mucoids  or  Mucinoids.  In  this  group  we  must  include  those  non- 
phosphorized  glucoproteids  which  are  neither  true  mucins  nor  chondro- 
proteids  even  though  they  show  amongst  themselves  such  a  difference  in 
behavior  that  they  can  be  divided  into  several  subgroups  of  mucoids. 
To  the  mucoids  belong  pseudomucin,  the  probably  related  body  colloid, 
ovomucoid,  and  other  bodies,  which  on  account  of  their  differences  will  be 
best  treated  individually  in  their  respective  chapters. 

Hyalogens.  Under  this  name  Krukenberg2  has  designated  a  number  of 
differing  bodies,  which  are  characterized  by  the  following:  By  the  action  of 
alkalies  they  change,  with  the  splitting  off  of  sulphur  and  some  nitrogen,  into 
soluble  nitrogenized  products  called  by  him  hyalines  and  which  yield  a  pure  car- 
bohydrate by  further  decomposition.  We  find  that  very  heterogeneous  substances 
are  included  in  these  groups.  Certain  of  these  hyalogens  seem  undoubtedly 
to  be  glucoproteids.  Neossin  3  of  the  Chinese  edible  swallow's-nest,  membranin  * 
of  Descemet's  membrane  and  of  the  capsule  of  the  crystalline  lens,  and  spiro- 
graphin*  of  the  skeletal  tissue  of  the  worm  Spirographis  seem  to  act  as  such. 
Others  on  the  contrary,  such  as  hyalin  8  of  the  walls  of  hydatid  cysts,  onuphin  ' 


1  The  author  has  not  been  able  to  obtain  this  pure,  so  the  analysis  is  not  given  in 
the  previous  table  of  the  mucins. 

:  Verh.  d.  physik.-med.  Gesellsch.  zu  Wiirzburg,  1883;  also  Zeitschr.  f.  Biologie,  22. 

3  Krukenberg,  Zeitschr.  f.  Biologie,  22. 

«C.  Th.  Morner,  Zeitschr.  f.  physiol.  Chem.,  18. 

5  Krukenberg,  Wiirzburg,  Verhandl.  1SS3;  also  Zeitschr.  f.  Biologie,  22. 

0  A.  Liicke,  Virchow's  Arch.,  19;  also  Krukenberg,  Vergleichende  physiol.  Stud.» 
Series  1  and  2,  1881. 

7  Schmiedeberg,  Mitth.  aus  d.  zool.  Stat,  zu  Neapel,  3,  1882. 


54  THE  PROTEIN  SUBSTANCES. 

from  the  tubes  of  Onuphis  tubicola,  do  not  seem  to  be  compound  proteids.  The 
so-called  mucin  of  the  holothures,1  and  chondrosin 2  of  the  sponge,  Chondrosia 
reniformis,  and  others  may  also  be  classed  with  the  hyalogens.  As  the  various 
bodies  designated  by  Krukenberg  as  hyalogens  are  very  dissimilar,  it  is  not 
of  much  importance  to  arrange  these  in  special  groups. 

3.  Chondroproteids  are  those  glucoproteids  which  as  closest  cleavage 
products  yield  proteid  and  an  ethereal  sulphuric  acid  containing  a  carbo- 
hydrate, chondroitin-sulphuric  acid.  Chondromucoid,  occurring  in  cartilage, 
is  the  best  example  of  this  group.  Amyloid  occurring  under  pathological 
conditions  also  belongs  to  this  group.  On  account  of  the  property  of  chon- 
droitin-sulphuric acid  of  precipitating  proteid  it  is  also  possible  that  under 
certain  circumstances  combinations  of  this  acid  with  proteid  may  be  pre- 
cipitated from  the  urine  and  be  considered  as  chondroproteids. 

The  chondromucoid,  the  so-called  tendon-mucin,  and  the  osseomucoid 
have  greatest  interest  as  constituents  of  cartilage,  of  the  connective  tissues, 
and  of  the  bones,  and  on  this  account  these  bodies  and  their  cleavage  prod- 
uct, chondroitin-sulphuric  acid,  will  be  treated  in  a  following  chapter  (X). 
On  the  contrary,  amyloid,  which  has  always  been  considered  in  connection 
with  the  protein  substances,  will  be  described  here. 

Amyloid,  so  called  by  Virchow,  is  a  protein  substance  appearing  under 
pathological  conditions  in  the  internal  organs,  such  as  the  spleen,  liver,  and 
kidneys  as  infiltrations;  and  in  serous  membranes  as  granules  with  con- 
centric layers.  It  probably  also  occurs  as  a  constituent  of  certain  prostate 
calculi.  The  chondroproteid  occurring  under  physiological  conditions  in 
the  walls  of  the  arteries  is  perhaps,  according  to  Krawkow,  very  nearly 
related  to  the  amyloid  substance  even  if  not  identical. 

Amyloid  was  first  prepared  pure  recently  by  Krawkow.  The  sub- 
stance prepared  by  him  contained  C  48.86-50.38;  H  6.65-7.02;  N  13.79- 
14.07;  and  S  2.65-2.89  per  cent.  Phosphorus  does  not  occur  in  the  pure 
substance.  It  splits,  by  the  action  of  alkali,  into  proteid  and  chondroitin- 
sulphuric  acid  (see  Chapter  X)  and  according  to  Krawkow  is  therefore 
perhaps  an  ester-like  combination  of  this  acid  with  proteid.  According 
to  Monery  3  amyloid  on  the  contrary  contains  phosphorus.  According 
to  him  the  proteid  component  is  a  nuclein  substance  and  the  other  com- 
ponent is  not  identical  with  chondroitin-sulphuric  acid. 

Amyloid  is  an  amorphous  white  substance,  insoluble  in  water,  alcohol, 
ether,  dilute  hydrochloric  and  acetic  acids.  It  is  soluble  in  concentrated 
hydrochloric  acid  or  caustic  alkali  with  decomposition.  On  boiling  with 
dilute  hydrochloric  acid  it  yields  sulphuric  acid  and  a  reducing  substance. 
It  is  not  dissolved  by  gastric  juice  according  to  Krawkow  and  most  older 

1  Hilger,  Pfliiger's  Archiv,  3. 

2  Krukenberg,  Zeitschr.  f.  Biologie,  22. 

3  Krawkow,  Arch.  f.  exp.  Path.  u.  Pharm.,  40,  which  contains  the  literature ;  Mon<5ry, 
Compt.  rend.  soc.  biol.,  54 


NUCLEOPROTEIDS.  55 

statements.  It  is  nevertheless  changed  so  that  it  is  soluble  in  dilute  ammo- 
nia, while  the  genuine  typical  amyloid  is  insoluble  therein.  Amyloid 
gives  the  xanthoproteic  reaction  and  the  reactions  of  Millon  and  Ada.u- 
ki i:\vicz.  Its  most  important  property  is  its  behavior  with  certain  coloring 
matters.  It  is  colored  reddish  brown  or  a  dingy  violet  by  iodine;  a  violet 
or  blue  by  iodine  and  sulphuric  acid;  red  by  methylaniline  iodide,  espe- 
cially on  the  addition  of  acetic  acid;  and  red  by  aniline  green.  Of  these 
color  reactions  those  with  aniline  dyes  are  the  most  important.  The  iodine 
reaction  appears  less  constant  and  is  greatly  dependent  upon  the  physical 
condition  of  the  amyloid.  The  color  reactions  are  dependent  upon  the 
presence  of  the  chondroitin-sulphuric  acid  component. 

The  preparation  of  amyloid  may  be  performed  as  follows  according 
to  Krawkow:  The  finely  divided  organ  is  exhausted  first  with  water 
and  then  with  dilute  ammonia,  which  leaves  the  insoluble  amyloid  and 
removes  the  free  or  the  combined  chondroitin-sulphuric  acid  besides  other 
substances.  The  product,  after  being  washed  with  water,  is  digested  with 
pepsin  for  several  days  at  38°  C.  The  residue,  after  washing  with  hydro- 
chloric acid  and  water,  is  dissolved  in  dilute  ammonia,  filtered,  again  pre- 
cipitated with  dilute  hydrochloric  acid,  dissolved,  if  necessary,  in  ammonia, 
precipitated  a  second  time  with  hydrochloric  acid,  washed  with  water,  the 
precipitate  dissolved  in  baryta-water,  which  leaves  the  nucleins  undis- 
solved, and  the  barium  filtrate  precipitated  with  hydrochloric  acid,  and 
then  washed  with  water,  alcohol,  and  ether. 

Phosphoglucoproteids.  This  group  includes  the  phosphorized  glucoproteids. 
They  yield  no  xanthine  substances  (nuclein  bases)  as  cleavage  products.  They 
are  not  nucleoproteids  and  therefore  they  must  not  be  considered  together 
with  the  gluconucleoproteids  (nucleoglucoprotcids)  or  mistaken  for  them.  On 
pepsin  digestion  they  may  like  certain  nucleoalbumins  yield  pseudonuclein, 
but  they  differ  from  the  nucleoalbumins  in  that  they  yield  a  reducing  substance 
on  boiling  with  dilute  acid.  They  differ  from  the  gluconucleoproteids  in  that 
they  do  not,  as  above  mentioned,  yield  any  xanthine  bodies. 

Only  two  phosphorized  glucoproteids  are  known  at  the  present  time,  namely, 
ichthulin,  occurring  in  carp  eggs  and  studied  by  Walter1  and  which  was  con- 
sidered as  a  vitellin  for  a  time.  Ichthulin  has  the  following  composition:  C  53.52; 
H  7.71;  N  15.64;  S  0.41;  P  0.43;  Fe  0.10  per  cent.  In  regard  to  solubilities  it 
is  similar  to  a  globulin.  "Walter  has  prepared  a  reducing  substance  from  the 
paranuclein  of  ichthulin  which  gave  a  very  crystalline  combination  with  phenyl- 
hydrazin. 

Another  phosphoglucoproteid  is  hclicoproteid,  obtained  by  Hammarstex  2 
from  the  glands  of  the  snail  Helix  pomatia.  It  has  the  following  composition: 
C  46.99;  H  6.78;  N  6.08;  S0.62;  P  0.47  per  cent.  It  is  converted  into  a  gummy, 
lspvorotatory  carbohydrate,  called  aninial  sinistrin,  by  the  action  of  alkalies 
On  boiling  with  an  acid  it  yields  a  dextrorotatory  reducing  substance. 

The  compound  proteid  found  by  Schulz  and  Ditthorn  3  in  the  pro- 
teid  glands  of  the  frog  probably  belongs  to  this  group,  but  it  does  not  yield 
glucosamine  but  gives  galactosamine  on  cleavage. 

1  Zeitschr.  f.  physiol.  Chem.,  15. 

2  Pfliiger's  Arch.,  30. 

3  Zeitschr.  f.  physiol.  Chem.,  29. 


56  THE  PROTEIN  SUBSTANCES. 


Nucleoproteids.  I  With  this  name  we  designate  those  compound  pro- 
teids  which  yield  true  nucleins  (see  Chapter  V)  on  pepsin  digestion  and 
on  treatment  with  dilute  caustic  alkali  yield  on  cleavage  proteid  and  nucleic 
acid^ 

The  nucleoproteids  seem  to  be  widely  diffused  in  the  animal  body. 
They  occur  chiefly  in  the  cell-nuclei,  but  they  also  often  occur  in  the  proto- 
plasm. They  may  pass  into  the  animal  fluids  on  the  destruction  of  the 
cells,  hence  nucleoproteids  have  also  been  found  in  blood-serum  and  other 
fluids. 

They  may  be  considered  as  combinations  of  a  proteid  nucleus  with  a  side 
chain,  which  Kossel  calls  the  prostetic  group.  This  side  chain,  which 
contains  the  phosphorus,  may  be  split  off  as  nucleic  acid  (see  Chapter  V)  on 
treatment  with  alkali.  As  we  have  several  nucleic  acids,  it  follows  that  we 
must  have  different  nucleoproteids,  depending  upon  the  nucleic  acid  united 
with  the  proteid.  Certain  nucleic  acids  contain  a  readily  split-off  sugar 
(pentose  or  hexose),  others,  on  the  contrary,  not.  In  the  first  case  we 
obtain  from  the  corresponding  nucleoproteid  a  reducing  sugar  on  boiling 
with  dilute  mineral  acid,  while  in  the  other  case  this  is  not  possible.  This 
different  behavior  may  be  accounted  for  by  a  special  group  of  nucleopro- 
teids, the  gluconucleoproteids  or  nucleoglucoproteids.  Such  gluconucleo- 
proteids  yielding  pentoses  occur  in  yeast-cells,  and,  as  it  appears,  are  widely 
distributed  in  the  animal  organism  (Blumenthal,  Grund  *). 

The  native  nucleoproteids  contain  a  variable  but  not  a  high  percentage 
of  phosphorus,  which  Halliburton  2  found  to  vary  between  0.5  per  cent 
and  1.6  per  cent.  On  heating  their  solutions,  as  well  as  by  the  action  of 
dilute  acids,  a  modification  of  the  compound  proteid  takes  place  and  nucleo- 
proteids of  strong  acid  character,  poorer  in  proteid  but  richer  in  phosphorus, 
are  formed.  The  native  nucleoproteids  have  faint  acid  properties  and  are 
insoluble  in  water,  but  their  alkali  combinations,  which  are  soluble  in  water, 
split  on  heating  their  solution  into  coagulated  proteid  and  a  nucleoproteid 
rich  in  phosphorus,  which  remains  in  solution.  In  peptic  digestion  they 
yield  so-called  true  nuclein,  which  is  also  a  nucleoproteid  poor  in  proteid. 
The  proteid  can  be  precipitated  by  acetic  acid  from  its  alkali  combination, 
and  the  precipitate  dissolves  with  more  or  less  readiness  in  an  excess  of 
the  acid.  A  confusion  may  occur  here  with  nucleoalbumins  and  also  with 
mucin  substances.  This  confusion  may  be  avoided  by  warming  the  body 
for  some  time  on  the  water-bath  with  dilute  sulphuric  acid,  nearly  neu- 
tralizing the  boiling-hot  fluid  with  barium  hydrate,  filtering  as  quickly  as 
possible  while  boiling  hot,  supersaturating  the  filtrate  with  ammonia,  and 

1  Blumenthal,  Berlin,  klin.  Wochenschr. ,  1897,  and  Zeitschr.  f.  klin.  Med.,  34; 
Grund,  Zeitschr.  f.  physiol.  Chem.,  35.  See  also  Bendix  and  Ebstein,  Zeitschr.  f. 
allgem.  Phys.,  2. 

3  Journ.  of  Physiol.,  18. 


KERA  TINS.  57 

then  on  cooling  (when  a  precipitate  consisting  of  guanine  is  filtered  off  and 
specially  tested)  testing  for  xanthine  bodies  by  an  ammoniacal  silver  nitrate 
solution.  Any  precipitate  formed  is  examined  more  closely  by  the  method 
as  given  in  Chapter  V.  The  nucleoproteids  give  the  color  reactions  of 
the  proteids,  but  those  which  have  been  investigated  are  dextrorotatory 
and  not  kevorotatory  (Gamgee  and  Jones  1). 

The  properties  of  the  various  nucleoproteids  are  given  in  detail  in  the 
various  chapters  which  follow. 

III.  Albumoids  or  Albuminoids. 

Under  this  name  we  collect  into  a  special  group  all  those  protein  bodies 
which  cannot  be  placed  in  either  of  the  other  two  groups,  although  they 
differ  essentially  among  themselves  and  from  a  chemical  standpoint  do  not 
show  any  radical  difference  from  the  true  proteid  bodies.  The  most  im- 
portant and  abundant  of  the  bodies  belonging  to  this  group  are  important 
constituents  of  the  animal  skeleton  or  the  cutaneous  structure.  They  occur 
as  a  rule  in  an  insoluble  state  in  the  organism,  and  they  are  distinguished 
in  most  cases  by  a  pronounced  resistance  to  reagents  which  dissolve  proteids 
or  to  chemical  reagents  in  general. 

The  Keratin  Group.  Keratin  is  the  chief  constituent  of  the  horny 
structure,  of  the  epidermis,  of  hair,  wool,  of  the  nail,  hoofs,  horns,  feathers, 
of  tortoise-shell,  etc.,  etc.  Keratin  is  also  found  as  neurokeratin  (Kuhne) 
in  the  brain  and  nerves.  The  shell-membrane  of  the  hen's  egg  seems 
also  to  consist  of  keratin,  and  according  to  Neumeister  2  the  organic 
matrix  of  the  egg-shells  of  various  vertebrate  animals  belongs  in  most 
cases  to  the  keratin  group. 

It  seems  that  there  exist  a  number  of  keratins,  and  these  form  a  special 
group  of  bodies.  This  fact,  together  with  the  difficulty  in  isolating  the 
keratin  from  the  tissues  in  a  pure  condition  without  a  partial  decomposi- 
tion, is  sufficient  explanation  for  the  variation  in  the  elementary  composition 
given  below.  As  examples  the  analyses  of  a  few  tissues  rich  in  keratin  and 
of  keratins  are  given  as  follows : 3 

C               H  N  SO 

Human  hair.  ..  .        50.65  6.36  17.14  5.00   20.85  (v.  Laar) 

Nail 51.00  6.94  17.51  2.80   21.75  (Mulder) 

Neurokeratin  .  .  .  56.11-58.45  7.26-8.02  11.46-14.32  1.63-2.24 (Kuhne) 

Horn  (average)..        50.86  6.94  3.20      (Horbaczewski) 

Tortoise-shell.  .  .        54.89  6.56  16.77  2.22    19.56  (Mulder) 

Shell-membrane.        49.78  6.94  16.43  4.25    22.90  (Lindvall) 

1  Hofmeister's  Beitriige,  4. 

2  Kuhne  and  Ewald,  Verh.  d.  naturhistor.-med.  Vereins  zu  Heidelberg  (N.  F.),  lj 
also  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  26;   Neumeister,  ibid.,  31. 

s  v.  Laar,  Annal.  d.  Chem.  u.  Pharm.,  45;  Mulder,  Versuch  einer  allgem.  physiol. 
Chem.,  Braunschweig,  1844-51;  Kuhne,  Zeitschr.  f.  Biologie,  26;  Horbaczewski,  see 
Drechsel  in  Ladenburg's  Handworterbuch  d.  Chem.,  3j  Lindvall,  Maly's  Jahres- 
bericht,  1881. 


58  THE  PROTEIN  SUBSTANCES. 

Mohr  *  has  determined  the  quantity  of  sulphur  in  various  keratin  sub- 
stances. Sulphur  is  in  great  part  in  loose  combination,  and  it  is  chiefly 
removed  by  the  action  of  alkalies  (as  sulp hides),  or  indeed  in  part  by  boiling 
with  water.  Combs  of  lead  after  long  usage  become  black,  and  this  is  due 
to  the  action  of  the  sulphur  of  the  hair.  On  heating  keratin  with  water  in 
sealed  tubes  to  a  temperature  of  150°  C.  or  higher,  it  dissolves,  with  the 
elimination  of  sulphuretted  hydrogen  or  mercaptan  (Bauer),  and  the 
solution  contains  proteose-like  substances  (Krukenberg)  and  called  atmid- 
keratin  and  atmidkeratose  by  Bauer.2  Keratin  is  dissolved  by  alkalies, 
especially  on  warming,  producing  besides  alkali  sulphides  also  proteose  sub- 
stances. 

Besides  the  well-known  cleavage  products  such  as  leucin,  aspartic  acid, 
glutamic  acid,  arginin,  and  lysin,  Fischer  and  Dorpinghaus  3  have  re- 
cently found  glycocoll,  alanin,  a-aminovalerianic  acid,  a-pyrolidin  car- 
bonic acid,  serin,  phenylalanin,  and  pyrrolidin  carbonic  acid  (secondary 
from  glutamic  acid)  among  the  cleavage  products  of  horn  substances. 
Emmerling  claims  to  have  found  cystin  as  a  sulphurized  cleavage  product, 
but  K.  Morner  4  has  recently  positively  proven  that  cystin  exists  as  an 
important  cleavage  product.  Morner  obtained  from  ox-horn,  human 
hair,  and  the  shell-membrane  of  the  hen's  egg  6.8,  13.92,  and  7.62  per 
cent  cystin  calculated  on  the  dry  substance.  From  the  amount  of 
sulphur  split  off  by  alkali,  he  concludes  that  at  least  in  ox-horn  and 
human  hair  all  the  sulphur  exists  as  cystin.  Suter,  and  after  him 
Friedmann,5  have  obtained  a-thiolactic  acid  as  a  hydrolytic  cleavage 
product  of  the  keratin  substances.  The  last-mentioned  investigator 
was  also  able  to  detect  thioglycolic  acid  in  the  cleavage  products  of 
wool. 

Bodies  occur  in  the  animal  kingdom  which  form  to  a  certain  extent 
intermediate  bodies  between  coagulated  proteid  and  keratin.  C.  Th. 
Morner  6  has  detected  such  a  body  {albumoid)  in  the  tracheal  cartilage, 
which  forms  a  net-like  trabecular  tissue.  This  substance  appears  to  be 
related  to  the  keratins  on  account  of  its  solubilities  and  the  quantity 
of  the  sulphur  (lead-blackening)  it  contains,  while  according  to  its 
solubility  in  gastric  juice  it  must  stand  close  to  the  proteids.  Another 
substance,  more  similar  to  keratin,  is  the  horny  layer  in  the  gizzard  of 


1  Zeitschr.  f.  physiol.  Chem.,  20. 

2  Krukenberg,    Untersuch.    iiber  d.    chem.  Bau   d.  Eiweisskorper.  Sitzungsber.    d. 
Jenaiscben   Gesellsch.  f.  Med.  u.  Naturwissensch. ,  1886;   Bauer,  Zeitschr.   f.    physiol. 

Chem.,  35. 

8  Zeitschr.  f.  physiol.  Chem.,  3G,  which  contains  also  the  older  literature. 

4  Morner,  ibid.,  34;  Emmerling,  Ref.  in  Chemiker  Zeitung,  1894. 

5  Suter,  Zeitschr.  f.  physiol.  Chem.,  20;  Friedmann,  Hofmeister's  Beitrage,  2. 

6  See  Maly's  Jahresber.,  18. 


ELASTIN.  59 

birds.  According  to  J.  Hedexius  l  this  substance  Is  insoluble  in  gastric 
or  pancreatic  juice  and  acts  quite  like  keratin.  It  contains  only  1  per 
cent  sulphur  and  yields  on  decomposition  only  very  little  tyrosin  besides 
considerable  leucin. 

Keratin  is  amorphous  or  takes  the  form  of  the  tissues  from  which  it  was 
prepared.  On  heating  it  decomposes  and  generates  an  odor  of  burnt  horn. 
It  is  insoluble  in  water,  alcohol,  or  ether.  On  heating  with  water  to  150°- 
200°  C.  it  dissolves.  It  also  dissolves  gradually  in  caustic  alkalies,  espe- 
cially on  heating.  It  is  not  dissolved  by  artificial  gastric  juice  or  by  tryp- 
sin solutions.  Keratin  gives  the  xanthoproteic  reaction,  as  well  as  the 
reaction  with  Millon's  reagent,  although  not  always  typical. 

In  the  preparation  of  keratin  a  finely  divided  horny  structure  is  treated 
first  with  boiling  water,  then  consecutively  with  diluted  acid,  pepsin- 
hydrochloric  acid,  and  alkaline  trypsin  solution,  and,  lastly,  with  water, 
alcohol,  and  ether. 

Elastin  occurs  in  the  connective  tissue  of  higher  animals,  sometimes  in . 
such  large  quantities  that  it  forms  a  special  tissue.      It  occurs  most  abun- 
dantly in  the  cervical  ligament  (ligamentum  nuchae). 

Elastin  used  to  be  generally  considered  as  a  sulphur-free  substance. 
According  to  the  investigations  of  Chittexdex  and  Hart,  it  is  a  question 
whether  or  not  elastin  does  not  contain  sulphur,  which  is  removed  by  the 
action  of  the  alkali  in  its  preparation.  H.  Schwarz  has  been  able  to 
prepare  an  elastin  containing  sulphur  from  the  aorta  by  another  method, 
and  this  sulphur  can  be  removed  by  the  action  of  alkalies,  without  changing 
the  properties  of  the  elastin,  and  recently  Zoja,  Hedix  and  Bergh,  Richards 
and  Gies  2  have  found  that  elastin  contains  sulphur.  The  most  trust- 
worthy analyses  of  elastin  from  the  cervical  ligament  (Xos.  1  and  2)  and 
from  the  aorta  (No.  3)  have  given  the  following  results,  which  seem  to 
compare  well  with  each  other. 

C  H  N          S          O 

1.  54.32  6.99  16.75  21.94  (Horbaczewski  3) 

2.  54.24  7.27  16.70  ....  21.79  (Chittenden  and  Hart) 

3.  53.96  7.03  10.67  0.38  (H.  Schwarz) 

Zoja  found  0.27G  per  cent  sulphur  and  16.96  per  cent  nitrogen  in  elastin. 
Hedix  and  Bergh  found  different  quantities  of  nitrogen  in  aorta-elastin, 
depending  upon  whether  IIorbaczewski 's  or  Schwarz  's  method  was 
used  in  its  preparation.     In  the  first  case  they  found  15.44  per  cent  nitro- 


1  Skan.  Arch.  f.  Physiol.,  3. 

2  Chittenden  and  Hart,  Zeitschr.  f.  Biologie,  25;  Schwarz,  Zeitschr.  f.  physiol. 
Chem.,  IS;  Zoja,  ibid.,  23;  Bergh,  ibid.,  2o;  Hedin,  ibid.;  Richards  and  Gies,  Amer. 
Journ.  of  Physiol.,  7. 

3  Horbaczewski,  Zeitschr.  f.  physiol.  Chem.,  6. 


60  THE  PROTEIN  SUBSTANCES. 

gen  and  0.55  per  cent  sulphur,  and  in  the  other  14.67  per  cent  nitrogen 
and  0.66  per  cent  sulphur.  Richards  and  Gies  found  0.14  per  cent  sulphur 
and  16.87  per  cent  nitrogen  in  elastin.  Abundant  leucin,  but  very  little 
tyrosin,  some  glycocoll,  and  perhaps  amino  valerianic  acid,  but  no  aspartic 
acid  or  glutamic  acid,  are  obtained  amongst  the  hydrolytic  cleavage  prod- 
ucts of  elastin.  The  three  hexon  bases  have  been  obtained,  but  only  in 
very  small  amounts,  so  that  the  basic  nitrogen  only  represents  3.34  per 
cent  of  the  total  nitrogen  (Richards  and  Gies).  This  fact  and  the  very 
low  sulphur  content  make  it  questionable  whether  the  elastin  is  a  unit 
body. 

On  putrefaction  by  anaerobic  micro-organisms  Zoja  found  carbon 
dioxide,  hydrogen,  methane,  mercaptan,  butyric  acid,  valerianic  acid, 
ammonia,  and  possibly  also  phenylpropionic  acid  and  aromatic  oxyacids. 
Indol  and  skatol  have  not  been  found  in  putrefaction,  but  Schwarz,1  on 
the  contrary,  obtained  indol,  skatol,  benzene,  and  phenols  on  fusing  aorta- 
elastin  with  caustic  potash.  On  heating  with  water  in  closed  vessels, 
on  boiling  with  dilute  acids,  or  by  the  action  of  proteolytic  enzymes,  the 
elastin  dissolves  and  splits  into  two  chief  products,  called  by  Horbac- 
zewski  hemielastin  and  elastinpeptone.  According  to  Chittenden  and 
Hart,  these  products  correspond  to  two  proteoses  designated  by  them 
protoelastose  and  deuteroelastose.  The  first  is  soluble  in  cold  water  and 
separates  on  heating,  and  its  solution  is  precipitated  by  mineral  acids  as 
well  as  by  acetic  acid  and  potassium  ferrocyanide.  The  water  solution  of 
the  other  does  not  become  cloudy  on  heating,  and  is  not  precipitated  by 
the  above-mentioned  reagents.  According  to  Richards  and  Gies  elastoses, 
especially  protoelastoses,  and  true  peptone  are  formed,  the  latter  only  to  a 
slight  extent. 

Pure  elastin  when  dry  is  a  yellowish-white  powder;  in  the  moist  state  it 
1  appears  like  yellowish-white  threads  or  membranes.  It  is  insoluble  in 
water,  alcohol,  or  ether,  and  shows  a  resistance  against  the  action  of 
chemical  reagents.  It  is  not  dissolved  by  strong  caustic  alkalies  at  the 
ordinary  temperature  and  only  slowly  at  the  boiling  temperature.  It  is 
very  slowly  attacked  by  cold  concentrated  sulphuric  acid,  and  it  is  relatively 
easily  dissolved  on  warming  with  strong  nitric  acid.  Elastins  of  differing 
origins  act  differently  with  cold  concentrated  hydrochloric  acid ;  for  instance, 
elastin  from  the  aorta  dissolves  readily  therein,  while  elastin  from  the 
ligamentum  nucha?,  at  least  from  old  animals,  dissolves  with  difficulty. 
Elastin  is  more  readily  dissolved  by  warm  concentrated  hydrochloric  acid. 
It  responds  to  the  xanthoproteic  reaction  and  with  Millon's  reagent. 

On  account  of  its  great  resistance  to  chemical  reagents,  elastin  may  be 
prepared  (best  from  the  ligamentum  nuchse)  in  the  following  way:    First 

1  See  Wiilchli,  Journ.  f.  prakt.  Chem.  (N.  F.),  17. 


COLLAGEN  S.  61 

boil  with  water,  then  with  1  per  cent  caustic  potash,  then  again  with  water, 
and  lastly  with  acetic  acid.  The  residue  is  treated  with  cold  5  per  cent 
hydrochloric  acid  for  twenty-four  hours,  carefully  washed  with  water, 
boiled  again  with  water,  and  then  treated  with  alcohol  and  ether. 

Tn  regard  to  the  methods  used  by  Schwarz,  Richards  and  Gies,  which  are 
somewhat  different,  we  refer  to  the  original  publication. 

Collagen,  or  gelatine-forming  substance,  occurs  very  extensively  in 
vertebrates.  The  flesh  of  cephalopoda  is  claimed  to  contain  collagen.1 
Collagen  is  the  chief  constituent  of  the  fibrils  of  the  connective  tissue  and 
(as  ossein)  of  the  organic  substances  of  the  bony  structure.  It  also  occurs 
in  the  cartilaginous  tissues  as  chief  constituent;  but  it  is  here  mixed  with 
other  substances,  producing  what  was  formerly  called  chondrigen.  Col- 
lagen  from  different  tissues  has  not  quite  the  same  composition,  and  prob- 
ably there  are  several  varieties  of  collagen. 

By  continuously  boiling  with  water  (more  easily  in  the  presence  of  a 
little  acid)  collagen  is  converted  into  gelatine.  Hofmeister  2  found  that 
gelatine  on  being  heated  to  130°  C.  is  again  transformed  into  collagen;  and 
this  last  may  be  considered  as  the  anhydride  of  gelatine.  Collagen  and 
gelatine  have  about  the  same  composition.3 

C  H         X  S  O 

Collagen 50.75  6.47  17.86  24.92  (Hofmeister) 

Gelatine  (commercial) 49.38  6.80  17.97  0.7       25.13  (Chittenden) 

Gelatine  from  tendons 50.11  6.56  17.81  0.26     25.26  (van  Name) 

Gelatine  from  ligaments.  .  50.49  6.71  17.90  0.57     24.33  (Richards  and  Gies) 

Fish  glue 48.69  6.76  17.68     (Faust) 

Gelatines  of  different  origin  show  a  somewhat  variable  composition, 
which  seems  to  indicate  the  occurrence  of  different  collagens.  It  is  diffi- 
cult to  say  whether  the  variable  content  of  sulphur  is  due  to  a  contam- 
ination with  a  substance  rich  in  sulphur  or  to  a  splitting  off  of  loosely  com- 
bined sulphur  during  the  purification.  C.  Morxer  *  has  prepared  a  typical 
gelatine  containing  only  0.2  per  cent  of  sulphur  by  a  method  which  elim- 
inated an}-  possible  changes  due  to  reagents. 

The  decomposition  products  of  the  collagens  are  the  same  as  those  of 
the  gelatines.  Besides  the  leucin,  glycocoll,  aspartic  acid  and  glutamic 
acid  found  by  the  earlier  investigators  as  hydrolytic  cleavage  products 
E.  Fischer  and  collaborators  5  have  obtained  alanin,  phenylalanin,  and 

1  Hoppe-Seyler,  Physiol.  Chem.,  97. 
:  Zeitschr.  f.  physiol.  Chem.,  2. 

3  Hofmeister,  1.  c. ;  Chittenden  and  Solley,  Journ.  of  Physiol.,  12;  Van  Name, 
Journ.  of  exper.  Med.,  2;  Richards  and  Gies,  Amer.  Journ.  of  Physiol.,  8;  Faust, 
Arch.  f.  exp.  Path.  u.  Pharm.,  41. 

4  Zeitschr.  f.  physiol.  Chem.,  28.      See  also  Sadikoff,  ibid.,  39. 

6  Fischer,  Levene  and  Aders,  Zeitschr.  f .  physiol.  Chem. ,  35.  In  regard  to  the 
older  researches,  see  O.  Cohnheim,  Chemie  die  Eiweisskrorper. 


62  THE  PROTEIN  SUBSTANCES. 

a-pyrroliclin  carbonic  acid.  Gelatine  does  not  give  any  tyrosin,  but  does 
yield  considerable  glycocoll  (16.5  per  cent  according  to  E.  Fischer),  which 
because  of  its  sweetish  taste  has  received  the  name  gelatine-sugar.  Gela- 
tine yields  considerable  basic  nitrogen,  according  to  Hausmann  *  35.83  per 
cent  of  the  total  nitrogen.  Drechsel  and  Fischer  found  lysin,  Hedin, 
Kossel  and  Kutscher  2  found  also  arginin,  which  amounted  to  9.3  per 
cent  (Kossel  and  Kutscher).  On  putrefaction  gelatine  gives  neither 
tyrosin,  indol,  nor  skatol.  According  to  Seltrenny  3  it  yields  phenyl- 
propionic  acid  and  phenylacetic  acid.  The  aromatic  group  in  gelatine  is 
therefore,  as  directly  shown  by  Fischer  (see  above)  and  also  by  Spiro,4 
represented  by  phenylalanin. 

Collagen  is  insoluble  in  water,  salt  solutions,  dilute  acids,  and  alkalies, 
but  it  swells  up  in  dilute  acids.  By  continuous  boiling  with  water  it  is 
converted  into  gelatine.  It  is  dissolved  by  the  gastric  juice  and  also  by  the 
pancreatic  juice  (trypsin  solution)  when  it  has  previously  been  treated  with 
acid  or  heated  with  water  above  70°  C.5  By  the  action  of  ferrous  sul- 
phate, corrosive  sublimate,  or  tannic  acid,  collagen  shrinks  greatly.  Col- 
lagen treated  by  these  bodies  does  not  putrefy,  and  tannic  acid  is  there- 
fore of  great  importance  in  the  preparation  of  leather. 

Gelatine  or  glutin  is  colorless,  amorphous,  and  transparent  in  thin  layers. 
It  swells  in  cold  water  without  dissolving.  It  dissolves  in  warm  water, 
forming  a  sticky  liquid,  which  solidifies  on  cooling  when  sufficiently  con- 
centrated. As  Pauli  and  Rona  6  have  shown,  various  bodies  may  have 
a  different  action  upon  the  gelatinization-point  of  a  gelatine  solution;  thus 
certain  bodies  such  as  sulphates,  citrates,  acetates,  and  glycerine  may 
accelerate,  while  the  chlorides,  chlorates,  bromides,  alcohol,  and  urea  retard 
this  power. 

Gelatine  solutions  are  not  precipitated  on  boiling,  neither  by  mineral 
acids,  acetic  acid,  alum,  basic  lead  acetate,  nor  metallic  salts  in  general.  A 
gelatine  solution  acidified  with  acetic  acid  may  be  precipitated  by  potassium 
ferrocyanide  on  carefully  adding  the  reagent.  Gelatine  solutions  are  precipi- 
tated by  tannic  acid  in  the  presence  of  salt;  by  acetic  acid  and  common 
salt  in  substance;  mercuric  chloride  in  the  presence  of  HC1  and  NaCl; 
metaphosphoric  acid,  phosphomolybdic  acid  in  the  presence  of  acid;  and 
lastly  also  by  alcohol,  especially  when  neutral  salts  are  present.  Gelatine 
solutions  do  not  diffuse.     Gelatine  gives  the  biuret  reaction,  but  not  Adam- 


1  Zeitschr.  f.  physiol.  Chem.,  27. 

2  Drechsel,  Arch.  f.  Anat.  u.  Physiol.,  1891;  Hedin,  Zeitschr.  f.  physiol.  Chem.,  21; 
Kossel  and  Kutscher,  ibid.,  31. 

3Monatsheft.  f.  Chem.,  10. 

4  Hofmeister's  Beitrage,  1. 

*Kiihne  and  Ewald,  Verh.  d.  Naturhist.  Med.  Vereins  in  Heidelberg,  1877,  1. 

•Hofmeister's  Beitrage,  2. 


GELATINE,   11ETICULIN.  63 

KiKwicz's.  It  gives  Millon's  reaction  and  the  xanthoproteic  reaction 
BO  faintly  that  it  probably  occurs  from  an  impurity  consisting  of  pro- 
teids.  According  to  C.  Morner,  pure  gelatine  gives  a  beautiful  Millon's 
reaction,  if  not  too  much  reagent  is  added.  In  the  other  case  no  reaction 
or  only  a  faint  one  is  obtained. 

By  continuous  boiling  with  water  gelatine  is  converted  into  a  non-gelat- 
inizing modification  called  /3-glutin  by  Nasse.  According  to  NASSE  and 
IEB  the  specific  rotatory  power  is  hereby  reduced  from  —  107. .V  to 
about  — 1360.1  On  prolonged  boiling  with  water,  especially  in  the  presence 
of  dilute  acids,  also  in  the  gastric  or  tryptic  digestion,  the  gelatine  is  trans- 
formed into  gelatine  proteoses,  so-called  gclatoscs  and  gelatine  peptones,  which 
diffuse  more  or  less  readily. 

According  to  Hofmkistkr  two  new  substances,  semiglutin  and  hemi- 
collin,  are  formed.  The  former  is  insoluble  in  alcohol  of  70-80  per  cent 
and  is  precipitated  by  platinum  chloride.  The  latter,  which  is  not  pre- 
cipitated by  platinum  chloride,  is  soluble  in  alcohol.  Chittenden  and 
Sollet  -  have  obtained  in  the  peptic  and  tryptic  digestion  a  proto-  and 
crogelatose,  besides  some  true  peptone.  The  elementary  composition 
of  these  gelatoses  does  not  essentially  differ  from  that  of  the  gelatine. 

According  to  Levexe  3  the  proto-  as  well  as  the  deuterogelatoses  yield 
a  lamer  amount  of  glycocoll,  even  20.3  per  cent,  than  the  gelatine  itself. 
Paal  4  has  prepared  gelatine  peptone  hydrochlorides  from  gelatine  by 
the  action  of  dilute  hydrochloric  acid.  These  salts  are  partly  soluble  in 
ethyl  and  methyl  alcohol,  and  partly  insoluble  therein.  The  peptones 
obtained  from  these  salts  contain  less  carbon  and  more  hydrogen  than 
the  gelatine  from  which  they  originated,  showing  that  hydration  has  taken 
place.  The  molecular  weigl\t  of  the  gelatine  peptone  as  determined  by 
Paal  by  Raoult's  method  was  200  to  3">2,  while  that  for  gelatine  was  S7S 
to  960.  The  gelatine  peptones  isolated  by  Siegfried  and  his  pupils 
ScHEERMESSER 6  and  Kruger  and  already  mentioned,  are  of  the  greatest 
inter 

Qagen  (contaminated  with  mucoid)  may -be  obtained  from  bones  by 
extracting  them  with  hydrochloric  acid  (which  dissolves  the  earthy  phos- 
phates) and  then  carefully  washing  the  acid  out  with  water.  It  may  be 
obtained  from  tendons  by  extracting  with  lime-water  or  dilute  alkali 
(which  dissolve  the  proteids  and  mucin)  and  then  thoroughly  washing  with 
water.  Gelatine  is  obtained  by  boiling  collagen  with  water.  The  finest 
commercial  gelatine  always  contains  a  little  proteid,  which  may  be  removed 

1  Xasse  and  Kruger,  Maly  s  Jahresber.,  19,  29.  In  regard  to  the  rotation  of  ^3-glutin 
see  Framm,  Pfliiger's  Arch.,  68. 

;  Bofmeister,  1.  c. ;   Chittenden  and  Solley,  1.  c. 

3  Levene,  Zeitschr.  f.  physiol.  Chem.,  3". 

4  Ber.  d.  deutsch.  chem.  Gesellsch..  25. 

5  Zeitschr.  f.  physiol.  Chem.,  37;   Kruger,  1.  c. 


64  THE  PROTEIN  SUBSTANCES. 

by  allowing  the  finely  divided  gelatine  to  swell  up  in  water  and  thoroughly 
extracting  with  large  quantities  of  fresh  water.  Then  dissolve  in  warm 
water  and  precipitate  with  alcohol. 

Collagen  may  also  be  purified  from  proteids  as  suggested  by  Van  Name 
by  digesting  with  an  alkaline  trypsin  solution  or  by  extracting  the  gelatine 
for  days  with  1-5  p.  m.  caustic  potash,  as  suggested  by  C.  Morner.  The 
typical  properties  of  gelatine  are  not  changed  by  this. 

Chondrin  or  cartilage  gelatine  is  only  a  mixture  of  gelatine  with  the  specific 
constituents  of  the  cartilage  and  their  transformation  products. 

Reticulin.  The  reticular  tissues  of  the  lymphatic  glands  contain  a 
variety  of  fibres  which  have  also  been  found  by  Mall  in  the  spleen, 
intestinal  mucosa,  liver,  kidneys,  and  lungs.  These  fibres  consist  of  a 
special  substance,  reticulin,  investigated  by  Siegfried.1 

Reticulin  has  the  following  composition:  C  52.88;  H  6.97;  N  15.63; 
S  1.88;  P  0.34;  ash  2.27  per  cent.  The  phosphorus  occurs  in  organic  combina- 
tion. It  yields  no  tyrosin  on  cleavage  with  hydrochloric  acid.  It  yields,  on 
the  contrary,  sulphuretted  hydrogen,  ammonia,  lysin,  arginin,  and  amino- 
valerianic  acid.  On  continuous  boiling  with  water,  or  more  readily  with 
dilute  alkalies,  reticulin  is  converted  into  a  body  which  is  precipitated  by 
acetic  acid,  and  at  the  same  time  phosphorus  is  split  off. 

Reticulin  is  insoluble  in  water,  alcohol,  ether,  lime-water,  sodium 
carbonate,  and  dilute  mineral  acids.  It  is  dissolved,  after  several  weeks, 
on  standing  with  caustic  soda  at  the  ordinary  temperature.  Pepsin-hydro- 
chloric acid  or  trypsin  do  not  dissolve  it.  Reticulin  responds  to  the  biuret, 
xanthoproteic,  and  Adamkiewicz 's  reactions,  but  not  with  Millon's 
reagent. 

According  to  Tebb  reticulin  is  only  a  somewhat  changed,  impure  collagen, 
but  this  is  disputed  by  Siegfried.2 

It  may  be  prepared  as  follows,  according  to  Siegfried:  Digest  intes- 
tinal mucosa  with  trypsin  and  alkali.  Wash  the  residue,  extract  with 
ether,  and  digest  again  with  trypsin  and  then  treat  with  alcohol  and  ether. 
On  careful  boiling  with  water  the  collagen  present  either  as  contamination 
or  as  a  combination  with  reticulin  is  removed.  The  thoroughly  dried 
residue  consists  of  reticulin. 

Ichthylepidin  is  an  organic  substance,  so  called  by  C.  Morner,3  which  occurs 
with  collagen  in  fish-scales  and  fomris  about  &  of  the  organic  substance  of  the  same. 
This  substance  with  15.9  per  cent  nitrogen  and  1.1  per  cent  sulphur  stands  on 
account  of  its  properties  rather  close  to  elastin.  It  is  insoluble  in  cold  and  hot 
water,  as  well  as  in  dilute  acids  and  alkalies  at  the  ordinary  temperature.     On 

'  Mall,  Abhandl.  d.  math.  phys.  Klasse  d.  Kgl.  sachs.  Gesellsch.  d.  Wiss.,  1891; 
Siegfried,  Ileber  die  chem.  eigensch.  der  reticulirten  Gewebe.  Habil.-Schrift.  Leipzig, 
1892. 

■  Tebb,  Journ.  of  Physiol.,  27;   Siegfried,  ibid.,  28. 

"  Zeitschr.  f.  physiol.  Chem.,  24  and  37.     See  also  Green  and  Tower,  ibid.,  35. 


SKELETINS.  65 

boiling  with  these  it  dissolves.  Pepsin-hydrochloric  acid,  as  well  as  an  alkaline 
trypsin  solution,  also  dissolves  it.  It  gives  beautiful  reactions  with  Mii.i.on's 
reagent,  xanthoproteic  reaction,  and  the  biuret  test.  At  least  a  part  of  the 
sulphur  is  split  off  by  the  action  of  alkali. 

Skeletins  are  a  number  of  nitrogenized  substances  which  form  the 
skeletal  tissue  of  various  classes  of  invertebrates  so  designated  by  Kru- 
KENBERG.1  These  substances  are  chitin,  s pong  in,  conchiolin,  corncin,  and 
fibroin  (silk).  Of  these  chitin  does  not  belong  to  the  protein  substances, 
and  fibroin  (silk)  is  hardly  to  be  classed  as  a  skeletin.  Only  those  so-called 
skeletins  will  be  given  that  actually  belong  to  the  protein  group. 

Spongin  forms  the  chief  mass  of  the  ordinary  sponge.  It  gives  no  gelatine. 
On  boiling  with  acids,  according  to  the  older  statements  it  yields  leucin  and 
glycocoll  and  no  tyrosin.  Zalocostas  claims  to  have  found  tyrosin  and  also 
butalanin  and  glucalanin  (C5H12X204).  After  Hundeshagen  had  shown  the 
occurrence  of  iodine  and  bromine  in  organic  combination  in  different  sponges  and 
designated  the  albumoid  containing  iodine,"  iodospongin,  Harnack  -  later  iso- 
lated from  the  ordinary  sponge,  by  cleavage  with  mineral  acids,  an  iodospongin 
which  contained  about  9  per  cent  iodine  and  4.5  per  cent  sulphur.  Conchiolin 
is  found  in  the  shells  of  mussels  and  snails  and  also  in  the  egg-shells  of  these  ani- 
mals. It  yields,  according  to  Wetzel,3  glycocoll,  leucin,  and  abundance  of  tyro- 
sin. The  quantity  of  diamino  nitrogen  amounts  to  8.7  per  cent  and  the  amid 
nitrogen  3.47  per  cent  (from  the  shell  of  pinna).  The  Byssus  contains  a  substance, 
closely  related  to  conchiolin,  which  is  soluble  with  difficulty.  Cornein  forms  the 
axial  system  of  the  Antipathes  and  Gorgonia.  It  gives  leucin  and  a  crystallizable 
substance,  comicryztallin.  According  to  Drechsel  the  axial  system  of  the  Gor- 
gonia cavolini  contains  nearly  8  per  cent  of  the  dry  substance  as  iodine.  The 
iodine  occurs  in  organic  combination  with  an  iodized  albumoid,  gorgonin,  which 
is  a  cornein.  Drechsel  obtained  leucin,  tyrosin,  lysin,  ammonia,  and  an  iodized 
amino  acid,  iodogorgonic  acid,  as  cleavage  products  of  gorgonin.  Henze  *  could 
only  obtain  this  acid  in  very  small  quantities,  and  by  acid  cleavage  of  gorgonin  he 
obtained  the  three  hexon  bases,  abundance  of  tyrosin,  and  very  little  leucin.  On 
cleavage  with  barium  hydrate  he  obtained  only  lysin  besides  tyrosin  and  glycocoll 
in  larger  amounts. 

Fibroin  and  sericin  are  the  two  chief  constituents  of  raw  silk.  By  the  action 
of  boiling  water  the  sericin  (silk  gelatine)  dissolves  and  can  be  obtained  by  a 
method  suggested  by  Boxm,5  while  the  more  difficultly  soluble  fibroin  remains 
undissolved  in  the  shape  of  the  original  fibre.  The  sericin,  whose  sufficiently 
concentrated"  hot  solution  gelatinizes  on  cooling,  is  precipitated  by  mineral  acids, 
several  metallic  salts,  and  by  acetic  acid  and  potassium  ferrocyanide.  As  cleavage 
products  E.  Fischer  and  Skita  obtained  alanin,  serin,  very  little  glycocoll, 
tyrosin,  arginin,  and  probably  also  lysin.  Leucin  had  been  found  previously. 
From  fibroin  they  obtained,  besides  the  previously  known  cleavage  products, 
glycocoll,  tyrosin,  and  alanin  (Weyl6),  also  leucin,  phenylalanin,  serin,  a-pyrroli- 

1  Grundziige  einer  vergl.  Physiol,  d.  thier.  Geriistsubst.     Heidelberg,  1885. 
-  Zalocostas,    Compt.  rend.,  10";    Hundeshagen,  Maly's  Jahresber.,  25;    Harnack, 
Zeitschr.  f.  physiol.  Chem.,  24. 

'Zeitschr.  f.  physiol.  Chem.,  29,  and  Centralbl.  f.  Physiol.,  13,  113. 

*  Drechsel,  Zeitschr.  f.  Biologie,  33;   Henze,  Zeitschr.  f.  physiol.  Chem.,  38. 
"Zeitschr.  f.  physiol.  Chem.,  34. 

•  Fischer  and  Skita,  ibid.,  33;  Fischer,  ibid.,  39;  Weyl,  Ber.  d.  d.  chem.  Gesellsch., 
21. 


66 


THE  PROTEIN  SUBSTANCES. 


din  carbonic  acid  (Fischer),  and  a  small  amount  of  arginin.  The  chief  products 
were  glycocoll,  36  per  cent,  alanin,  21  per  cent,  and  tyrosin,  10  per  cent.  The 
composition  of  the  above-mentioned  albuminoids  is  as  follows:1 


C  H  N 

Conchiolin  (from  the  shells  of  pinna).  .  52.70  6.54  16.60 

(from  snail-eggs) 50.92  6.88  17.86 

Spongin 46.50  6.30  16.20 

48.75  6.35  16.40 

Cornein 48.96  5.90  16.81 

Fibroin 48.23  6.27  18.31 

"      .'...48.30  6.50  19.20 

Sericin 44.32  6.18  18.30 

44.50  6.32  17.14 


S 
0.85 
0.31 
0.50 


(Wetzel) 

(Krukenberg) 

(Croockewitt) 

(Posselt) 

(Krukenberg) 

(Cramer) 

(Vignon) 

(Cramer) 

(BONDl) 


APPENDIX   TO  CHAPTER  II. 
HYDROLYTIC    CLEAVAGE   PRODUCTS  OF   THE   PROTEIN   SUBSTANCES. 

i.  Monamino  Acids. 

Glycocoll  (aminoacetic   acid),  C2H5N02=CH2(.NH2),  also  called  glycin 

COOH 
or  gelatine  sugar,  is  found  in  the  muscles  of  the  Pecten  irradians,  but 
has  chief  interest  as  a  hydrolytic  decomposition  product  of  protein  bodies, 
especially  gelatine,  fibroin,  and  spongin,  as  well  as  of  hippuric  acid  and 
glycocholic  acid.  It  is  also  found  in  the  decomposition  of  uric  acid, 
xanthine,  guanine,  and  adenine. 

The  largest  amounts  of  glycocoll  obtained  thus  far  from  the  protein 
substances  was  from  fibroin  2  (36  per  cent),  gelatine,  and  gelatoses  3  (16.5 
and  20.3  per  cent  respectively). 

Glycocoll  forms  colorless,  often  large,  hard  rhombic  crystals  or  four- 
sided  prisms.  The  crystals  have  a  sweet  taste  and  dissolve  readily  in 
cold  water  (4.3  parts).  It  is  insoluble  in  alcohol  and  ether  and  dissolves 
with  difficulty  in  warm  alcohol.  Glycocoll  combines  with  acids  and  alkalies. 
Among  the  latter  compounds  we  must  mention  those  with  copper  and 
silver.  Glycocoll  dissolves  cupric  hydrate  in  alkaline  liquids  but  does 
not  reduce  at  boiling  heat.  A  boiling-hot  solution  of  glycocoll  dissolves 
freshly  precipitated  cupric  hydrate,  forming  a  blue  solution,  which,  in 
proper  concentration,  deposits  blue  needles  of  glycocoll-copper  on  cooling. 
The  combination  with  hydrochloric  acid  is  readily  soluble  in  water  but 
less  soluble  in  alcohol. 


1  Krukenberg,  Ber.  d.  d.  chem.  Gesellsch.,  17  and  IS,  and  Zeitschr.  f.  Biologie,  22; 
Croockewitt,  Annal.  d.  Chem.  u.  Pharm.,  48;  Posselt,  ibid.,  45;  Cramer,  Journ.  f. 
prakt.  Chem.,  90;   Vignon,  Compt.  rend.,  115;    Wetzel,  1.  c,  and  Bondi,  1.  c. 

2  E.  Fischer  and  Skita,  Zeitschr.  f.  physiol.  Chem.,  33. 

3  E.  Fischer,  Levene  and  Aders,  ibid.,  35;   Levenc,  ibid.,  37. 


ALANIN.  07 

It  is  not  precipitated  even  in  a  5  per  cent  solution  by  phosphotungstie 
arid.  By  the  action  of  gaseous  HC1  upon  glycocoll  in  absolute  alcohol,  beau- 
tiful crystals  arc  obtained  of  the  hydrochloride  of  glycocoll  ethyl  ester  which 
melts  at  144°  C,  and  from  which  the  glycocoll  ethyl  ester  can  be  obtained 
by  the  method  suggested  by  E.  Fischer  '  for  the  separation  of  glycocoll 
from  the  other  amino  acids.     On  shaking  with  benzoyl  chloride  and  caustic 

hippuric  acid  is  funned,  and  this  is  also  made  use  of  indifferent 
in  detecting  and  isolating  glycocoll  (Ch.  Fischer,  GoNNERMANN,  SPIRO3) 

Glycocoll  can  be  best  prepared  from  hippuric  acid  by  boiling  it  with 
4  parts  dilute  sulphuric  acid  (1:0)  for  ten  to  twelve  hours.  After  cooling 
the  benzoic  acid  is  removed,  the  filtrate  concentrated,  the  remaining  benzoic, 
acid  removed  by  extracting  with  ether,  the  sulphuric  acid  precipitated  by 
BaC03,  and  the  filtrate  evaporated  to  point  of  crystallization.  (In  regard 
to  its  preparation  from  protein  substances  see  below.) 

CH3 

Alanin  (a-aminopropionic  acid),  C3H7NO,  =  CH(XH2),  was  first  obtained 

COOH 
by  Weyl  as  a  cleavage  product  of  fibroin.     This  alanin,  the  d-alanin,  has 
been  isolated  by  E.  Fischer  and  his  collaborators  3  more  abundantly  from 
fibroin  (21  per  cent)  and  also  from  sericin  (5  per  cent),  horn  substance 
(1.20  per  cent),  gelatine  (0.8  per  cent),  and  haemoglobin  (2.87  per  cent). 

Alanin  has  a  sweet  taste,  is  readily  soluble  in  water,  and  dissolves  cupric 
hydrate  on  boiling,  producing  alanin-copper,  which  has  a  deep-blue  color. 
The  specific  rotation  of  the  hydrochloride  (9-10  per  cent  solution)  is  (o:)d  = 
+  9. 0S°.  In  regard  to  the  synthetical  preparation  of  i-alanin,  its  cleavage 
as  benzoyl  compound,  and  the  preparation  of  i-alanin  ethyl  ester  we  must 
refe    to  E.  Fischer.4 

CH2OH 

Serin   (a-amino-?-oxypropionic  acid),  C3H7NO,  =  CH(NH2),  was  obtained  by 

COOH 
E.  Fischer  and  his  collaborators5  as  a  cleavage  product  from  fibroin  (1.6  per  cent), 
horn   substance    (0.68   per   cent),  sericin,  gelatine,  and   casein.     Synthetically  it 
was  prepared  by  E.  Fischer  and  Leuchs"  from  ammonia,  hydrocyanic  acid, 
and  glycol  aldehyde. 

It  does  not  dissolve  readily  in  cold  water  (23  parts  water  at  20°  C.)  but  more 


1  Ber.  d.  d.  chem.  Gesellsch.,  34. 

2  Ch.  Fischer,  Zeitschr.  f.  physiol.  Chem.,  19;  Spiro,  ibid.,  28;  Gonnermann, 
Pfliiger's  Arch.,  59. 

3  Weyl,  Ber.  d.  d.  chem.  Gesellsch.,  21;  Fischer  and  Skita,  Zeitschr.  f.  physiol. 
Chem.,  33;  Fischer  and  Dorpinghaus,  ibid.,  36;  Fischer,  Levene  and  Aders,  ibid., 
35     Fischer  and  Abderhalden,  ibid.,  36. 

4  Ber.  d.  d.  chem.  Gesellsch.,  32  and  31. 
sSee  foot-note  5,  page  66. 

•Ber.  d.  d.  chem.  Gesellsch.,  35,  and  Sitz.  Ber.  d.  Akad.  d.  Wiss.     Berlin,  1902. 


6S  THE  PROTEIN  SUBSTANCES. 

easily  in  hot  water.     The  solution  is  inactive   and  has  a  sweet  taste.    Serin  crys- 
tallizes from  water  in  thin  plates,  which  melt  at  240°  with  the  generation  of  a  gas. 

CHoCH, 


CH 
Aminovalerianic  acid,  C5HuN02=      CH(NH2),  has  been  detected  several  times 

COOH 
among  the  cleavage  products  of  protein  substances.     The  acid  isolated    by  E. 
Fischer  from  horn  substance  (5.70  per  cent)  and  casein,  as  well  as  that  obtained 
by  Schulze  and  Winterstein  x  from  lupin  sprouts,  seems  to  be  dextrorotatory 
a-aminovalerianic  acid. 

Leucin   (aminocaproic  acid,  or,  more   correctly,  a-aminoisobutylacetic 

CH3  CH3 

\/ 
CH 

acid,    C6H13N02  =     CH2  ,   is  produced   from    protein    substances    in 

CH(NH2) 

COOH 
their  hydrolytic  cleavage  by  proteolytic  enzymes  or  by  boiling  with  dilute 
acids  or  alkalies  or  by  fusing  with  alkali  hydrates  and  by  putrefaction. 
Because  of  the  ease  with  which  leucin  and  tyrosin  are  formed  in  the  decom- 
position of  protein  substances,  it  is  difficult  to  positively  decide  whether 
these  bodies  when  found  in  the  tissues  are  constituents  of  the  living  body 
or  are  only  to  be  considered  as  decomposition  products  formed  after  death. 
Leucin  it  seems  has  been  found  as  a  normal  constituent  of  the  pancreas 
and  its  secretion,  in  the  spleen,  thymus,  and  lymph  glands,  in  the  thyroid 
gland,  in  the  salivary  glands,  in  the  kidneys  and  liver.  It  also  occurs  in 
the  wool  of  sheep,  in  dirt  from  the  skin  (inactive  epidermis),  and  between 
the  toes,  and  its  decomposition  products  have  the  disagreeable  odor  of 
the  perspiration  of  the  feet.  It  is  found  pathologically  in  atheromatous 
cysts,  ichthyosis  scales,  pus,  blood,  liver,  and  urine  (in  diseases  of  the 
liver  and  phosphorus  poisoning).  Leucin  occurs  often  in  invertebrates 
and  also  in  the  plant  kingdom.  On  hydrolytic  cleavage  various  protein 
substances  yield  different  amounts  of  leucin.  Erlenmeyer  and  Schoffer 
obtained  36-45  per  cent  leucin  from  the  cervical  ligament,  Cohn  32  per 
cent  from  casein,  and  Nencki  1.5-2  per  cent  from  gelatine.  E.  Fischer 
and  Abderhalden  obtained  20  per  cent  leucin  from  haemoglobin,  Fischer 
and  Dorpinghaus  18.3  per  cent  from  horn  substance,  and  Fischer  and 
Skita  1.5  per  cent  from  fibroin.2 


1  Fischer,  Zeitschr.  f.  physiol.  Chem.;  36  and  33;  Schulze  and  Winterstein;  ibid.,  35. 

1  Erlenmeyer  and  Schoffer,  cited  from  Maly,  Chem.  d.  Verdauungssiifte,  in  Her- 
mann's Handb.  d.  Physiol.,  5,  Theil  2,  p.  209;  Cohn,  Zeitschr.  f.  physiol.  Chem.,  22; 
Nencki,  Journ.  f.  prakt.  Chem.  (N.  F.),  15;  Fischer  and  his  collaborators,  see  page  66, 
foot-note  5. 


LEUCIN.  69 

Leucin  occurs,  like  other  monamino  acids,  in  the  1-,  d-,  and  i-modifica- 
tions.  The  leucin  obtained  by  cleavage  of  protein  substances  is  generally 
the  form  which  is  dextrorotatory  in  acid  or  alkaline  solutions.  The 
leucin  prepared  synthetically  by  Hufner1  from  Lsovalemldehyde,  ammonia, 
and  hydrocyanic  acid  is  optically  inactive.  Inactive  leucin  may  also 
be  prepared,  as-  shown  by  E.  Schulze  and  Bosshard,2  by  the  cleavage 
of  proteids  with  baryta  at  160°  C.  or  on  heating  ordinary  leucin  with 
baryta-water  to  the  same  temperature.  The  lsevorotatory  modification 
may  be  formed  from  the  inactive  leucin  by  the  action  of  penicillum 
glaucum.  On  benzoylating  i-leucin  we  obtain  i-benzoyl-leucin,  from  whose 
cinchonine  and  quinidine  salts  first  d-  and  then  1-benzoyl-leucin  are  pre- 
pared and  then  by  hydrolytic  cleavage  d-  and  1-leucin  may  be  obtained 
(E.  Fischer).  On  oxidation  the  leucins  yield  the  corresponding  oxyacids 
(leucinic  acids).  Leucin  is  decomposed  on  heating,  evolving  carbon 
dioxide,  ammonia,  and  amylamine.  On  heating  with  alkalies,  as  also  in 
putrefaction,  it  yields  valerianic  acid  and  ammonia. 

Leucin  crystallizes  when  pure  in  shining,  white,  very  thin  plates,  usually 
forming  round  knobs  or  balls,  either  appearing  like  hyalin  or  alternating 
light  or  dark  concentric  layers  which  consist  of  radial  groups  of  crystals. 
On  slowly  heating  they  melt  at  170°  C.  and  sublime  in  white,  woolly  flakes, 
which  are  similar  to  sublimed  zinc  oxide.  At  the  same  time  an  odor  of 
amylamine  is  developed. 

Leucin  as  obtained  from  animal  fluids  and  tissues  is  very  easily  soluble 
in  wrater  and  rather  easily  in  alcohol.  Pure  leucin  is  soluble  with  difficulty. 
Pure  1-  and  d-leucin  dissolve  in  40-46  parts  wTater,  more  readily  in  hot 
alcohol,  but  with  difficulty  in  cold  alcohol.  The  i-leucin  is  much  less  soluble. 
According  to  Habermann  and  Ehrenfeld  3  100  parts  of  boiling  glacial 
acetic  acid  dissolve  29.93  parts  of  leucin.  The  specific  rotation  of  the 
ordinary  leucin,  dissolved  in  hydrochloric  acid,  is  about  (o:)d=  +  17.5. 

The  solution  of  leucin  in  water  is  not,  as  a  rule,  precipitated  by  metallic 
salts.  The  boiling-hot  solution  may,  however,  be  precipitated  by  a  boiling- 
hot  solution  of  copper  acetate,  and  this  is  made  use  of  in  separating  leucin 
from  other  substances.  If  the  solution  of  leucin  is  boiled  with  sugar  of 
lead  and  then  ammonia  be  added  to  the  cooled  solution,  shining  crystalline 
leaves  of  leucin-lead  oxide  separate.  Leucin  dissolves  cupric  hydrate, 
but  does  not  reduce  on  boiling. 

Leucin  is  readily  soluble  in  alkalies  and  acids.  It  gives  crystalline  com- 
pounds with  mineral  acids.  If  leucin  hydrochloride  is  boiled  with  alcohol 
containing  3-4  per  cent  HC1,  long  narrow  crystalline  prisms  of  hydrochloric- 
acid    leucin    ethyl    ester    melting    at    134°  C.  are    formed    (Rohmaxx). 

1  Joum.  f.  prakt.  Chem.  (N.  F.),  1. 

3  See  Zeitschr.  f.  physiol.  Chem.,  9  and  10. 

3  Zeitschr.  f.  physiol.  Chem.,  37. 


70  THE  PROTEIN  SUBSTANCES. 

The  same  is  produced  by  the  action  of  gaseous  HC1  upon  leucin  and  alcohol 
and  the  free  ethyl  ester  can  be  obtained  from  this  by  the  method  suggested 
by  E.  Fischer.1  This  ester  can  be  separated  from  the  other  amino-acid 
esters  by  distillation.  The  leucin  can  be  prepared  from  the  ester  by  boil- 
ing with  water  for  a  long  time.  The  picrate  of  the  leucin  ester  melts  at 
128°  C.  The  phenylisocyanate  compound  of  i-leucin  melts  at  165°  C. 
and  its  anhydride  at  125°  C. 

Leucin  is  recognized  by  the  appearance  of  balls  or  knobs  under  the 
microscope,  by  its  action  when  heated  (sublimation  test),  and  by  its 
compounds,  especially  the  hydrochloride  and  picrate  of  the  ethyl  ester 
and  the  phenylisocyanate  compound  of  the  racemic  leucin  obtained  by 
heating  with  baryta-water.  Leucin  must  first  be  isolated  before  it  can 
be  detected,  and  this  is  best  done  by  the  preparation  of  the  ethyl  ester  and 
then  distilling  it. 

^  TT  AT  ^     QH9.CH.NH.CO  n         _  .     .  .      „ 

Leucinimid,  C^H^A^Og  =  ^  Wu  prr  p  n  ,  was  first  obtained  by  Ritt- 

hattsen  in  the  hydrolytic  cleavage  products  on  boiling  proteids  with  acids  and 
subsequently  by  R.  Cohn.  Salaskin  2  obtained  it  in  the  peptic  and  tryptic 
digestion  of  haemoglobin.  It  may  probably  be  formed  as  anhydride  of  leucin 
(2.5  diacipiperazine)  by  a  secondary  change  from  leucin. 

It  crystallizes  in  long  needles  and  sublimes  readily.  The  melting-point  has 
not  been  found  constant  in  the  different  cases.  The  leucinimid  (3.6-di-isobutyl- 
2.5  diacipiperazine)  prepared  synthetically  by  E.  Fischer3  from  leucin  ethyl  ester 
melted  at  271°  C. 

COOH 

CTIYNTT  ^ 
Aspartic   Acid    (aminosuccinic    acid),    C4H7N04=^TT  ,   has    been 

COOH 

obtained  on  the  cleavage  of  protein  substances  by  proteolytic  enzymes 
as  well  as  by  boiling  them  with  dilute  mineral  acids.  Hlasiwetz  and 
Habermanst  obtained  23.8  per  cent  from  ovalbumin  and  9.3  per  cent 
from  casein,  although  not  quite  pure.  E.  Fischer  and  co-workers  4  ob- 
tained 3.29  per  cent  aspartic  acid  from  haemoglobin,  2.50  per  cent 
from  horn  substance,  and  0.56  per  cent  from  gelatine.  This  acid  also 
occurs  in  secretions  of  sea-snails  (Henze  5)  and  is  very  widely  diffused 
in  the  vegetable  kingdom  as  the  amid  Asparagin  (aminosuccinic-acid  amid), 
which  seems  to  be  of  the  greatest  importance  in  the  development  and 
formation  of  the  proteids  in  the  plants. 

1  Rohmann,  Ber.  d.  d.  chem.  Gesellsch.,  30;   E.  Fischer,  ibid.,  34. 

2  Ritthausen,  Die  Eiweisskorper  der  Getreidearten,  etc.,  Bonn,  1872j  R.  Cohn, 
Zeitschr.  f.  physiol.  Chem.,  22  and  29;  Salaskin,  ibid.,  32. 

3  Ber.  d.  d.  chem.  Gesellsch.,  34. 

4  Hlasiwetz  and  Habermann,  Annal.  d.  Chem.  u.  Pharm.,  159  and  169 j  E.  Fischer 
and  collaborators,  see  foot-note  5,  page  66. 

5  Ber.  d.  d.  chem.  Gesellsch.,  34. 


GLUTAMIC   ACID.  <1 

Aspartic  acid  dissolves  in  266  parts  water  at  10°  C.  and  in  18.6  parte 
boiling  water,  and  crystallizes  on  cooling  as  rhombic  prisms.  The  acid 
prepared  from  protein  substances  Ls  optically  active,  and  its  1  per  cent  solu- 
tion acidified  with  BC1  has  a  rotation  (a)D= +25.7°  and  dextrogyrate  or 

tevogyrate  in  a  watery  solution,  depending  upon  the  temperature.  It 
forms  with  copper  oxide  a  crystalline  combination  which  Ls  soluble  in  boiling- 
hot  water  and  nearly  insoluble  in  cold  water,  and  which  may  be  used  in 
the  preparation  of  the  pure  acid  from  a  mixture  with  other  bodies. 

In  regard  to  the  benzoylaspartic  acids  and  the  diethylester  we  must 
refer  to  the  work  of  E.  Fischeb  and  his  collaborators.  For  the  detection 
we  make  use  of  the  analysis  of  the  free  acid  and  the  copper  salts  as  wel- 
as  the  specific  rotation. 

COOH 
CH(NH2) 
Glutamic  acid  (a-aminoglutaric  acid),  C5H9N04=CH2  ,  is  obtained 

CH2 
COOH 
from  the  protein  substances  under  the  same  conditions  as  the  other  mon- 
amino  acids  and  from  the  peptones  (Siegfried).     Hlasiwetz  and  Haber- 
maxx  obtained  29  per  cent  from  casein  by  cleavage  with  hydrochloric  acid 
while  Kutscher  could  only  obtain  l.S  per  cent  glutamic  acid  by  cleavage 
with  sulphuric  acid.     Horbaczewski  has  obtained  15-18  per    cent  glu- 
tamic acid  from   gelatine  and   about  the   same   amount  from  horn,  while 
Fischeb  and  Dorpixghaus  obtained  only  3  per  cent  from  horn.     Fischer 
and  Ar.DERHALDEX  obtained  1.06  percent  from  haemoglobin,  and  Kutscher  ' 
3.66  per  cent  from  thymus  histon. 

Glutamic  acid  crystallizes  in  rhombic  tetrahedra  or  octahedra  or  in 
small  leaves.  It  melts  at  i:;.~>-140°  C.  with  partial  decomposition.  It  dis- 
solves in  100  parts  water  at  16°  C,  and  in  1500  parts  SO  per  cent  alcohol.  It 
is  insoluble  in  alcohol  and  ether.  The  d-glutamic  acid  obtained  from  pro- 
teids  by  boiling  with  an  acid  is  dextrorotatory;  a  5  per  cent  solution  of 
glutamic  acid  containing  9  per  cent  HC1  has  a  rotation  (o:)d=  +31.7°, 
while  that  obtained  by  heating  with  barium  hydrate  is  optically  inactive. 
The  d-glutamic  acid  forms  a  beautifully  crystalline  combination  with  hydro- 
chloric acid,  which  is  nearly  insoluble  in  concentrated  hydrochloric  acid. 
This  combination  Ls  used  in  the  isolation  of  glutamic  acid.  On  boiling  with 
cupric  hydrate  a  beautiful  crystalline  copper  salt,  which  is  soluble  with  diffi- 
culty, is  obtained.  Like  the  monamino  acids  in  general,  glutamic  acid  is 
not  precipitated  by  phosphotungstic  acid.  In  regard  to  the  benzoylglu- 
tamic  acids  and  the  diethylester  we  must  refer  to  the  works  of  Fischer.2 

1  Hlasiwetz  and    Hahcrmann.  1.  c,  159;    Kutscher,  Zeitschr.  f.  physiol.  Chem.,  28 
and  3S;   Ilorhaczewski,  Malvs  Jahres.,  10;   Fischer  and  collaborators,  1.  c. 
»L.  c 


72  THE  PROTEIN  SUBSTANCES. 

The  hydrochloride,  the  analysis  of  the  free  acid,  and  the  specific  rotation', 
are  used  in  its  detection. 

C6H,(OH) 
CH2 
Tyrosin  (p-oxyphenyl-a-aminopropionic  acid),  C9HuN03=CH(NH2),    is 

COOH 

produced  from  most  protein  substances  (not  from  gelatine  and  reticulin) 
under  the  same  conditions  as  leucin,  which  it  habitually  accompanies.  The- 
largest  quantity  of  tyrosin  obtained  from  animal  proteids  was  obtained 
by  Fischer  and  Skita  from  fibroin,  namely,  10  per  cent.  The  maxi- 
mum obtained  from  thymus  histon  (Kutscher)  was  6.3  per  cent,  from 
horn  substance  (R.  Cohn)  4.6  per  cent,  from  casein  (Reach)  4.55  per  cent, 
from  fibrin  (Kuhne)  3.86  per  cent,  from  ovalbumin,  seralbumin,  and  ser- 
globulin  (K.  Morner)  2.4,  2.0,  and  3.0  per  cent  respectively,  from  syntonin 
(Reach)  1.37  per  cent,  from  haemoglobin  (Fischer  and  Abderhalden) 
1.5  per  cent,  and  from  elastin  (Schwarz  *)  0.34  per  cent.  It  is  especially 
found  with  leucin  in  large  quantities  in  old  cheese  (Tvpos),  from  which 
it  derives  its  name.  Tyrosin  has  not  with  certainty  been  found  in  per- 
fectly fresh  organs.  It  occurs  in  the  intestine  in  the  digestion  of  proteid 
substances,  and  it  has  about  the  same  physiological  and  pathological  im- 
portance as  leucin. 

Tyrosin  was  prepared  by  Erlenmeyer  and  Lipp  from  p-aminophenyl- 
alanin  by  the  action  of  nitrous  acid,  and  according  to  another  method  by 
Erlenmeyer  and  Halsey.2  On  fusing  with  caustic  alkali  it  yields  p-oxy- 
benzoic  acid,  acetic  acid,  and  ammonia.  On  putrefaction  it  may  yield 
p-hydrocoumaric  acid,  oxyphenylacetic  acid,  and  p-cresol. 

Naturally  occurring  tyrosin  and  that  obtained  by  the  cleavage  of  protein 
substances  is  generally  1-tyrosin,  while  that  obtained  by  decomposition  with, 
baryta-water  or  prepared  synthetically  is  i-tyrosin.  v.  Lippmann  3  has 
obtained  d-tyrosin  from  beet-sprouts.  The  specific  rotation  of  ordinary 
tyrosin  dissolved  in  21  per  cent  hydrochloric  acid  varies  somewhat; 
(a)D= +7.98  and  8.64°. 4 

Tyrosin  in  a  very  impure  state  may  be  in  the  form  of  balls  similar  to 
leucin.     The  purified  tyrosin,  on  the  contrary,  appears  as  colorless,  silky, 

1  Fischer  and  Skita,  1.  c. ;  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  38;  R.  Cohn,  ibid.,. 
26;  Reach,  Virchow's  Arch.,  158;  Kuhne,  ibid.,  39;  K.  Morner,  Zeitschr.  f.  physiol. 
Chem.,  34;  Fischer  and  Abderhalden,  ibid.,  1  c. ;  Schwarz,  ibid.,  18. 

2  Erlenmeyer  and  Lipp,  Ber.  d.  d.  chem.  Gesellsch.,  15;  Erlenmeyer  and  Halsey t. 
ibid.,  30. 

3  Ibid.,  17. 

4  See  Hoppe-Seyler-Thierf  elder,  Handb.  d.  physiol.  u.  pathol.  Chem.  Analyse,  7~ 
Auflage,  1903. 


PHENYLALANIN.  73 

fine  needles  which  are  often  grouped  into  tufts  or  balls.  It  is  soluble  with, 
difficulty  in  water,  being  dissolved  by  2454  parts  water  at  20°  C.  and  154 
parts  boiling  water,  separating,  however,  as  tufts  of  needles  on  cooling.  It 
dissolves  more  easily  in  the  presence  of  alkalies,  ammonia,  or  a  mineral  acid. 
It  is  difficultly  soluble  in  acetic  acid.  Crystals  of  tyrosin  separate  from  an 
ammoniacal  solution  on  the  spontaneous  evaporation  of  the  ammonia.  One 
hundred  parts  glacial  acetic  acid  dissolve  on  boiling  only  0.18  parts  tyrosin, 
and  by  this  means,  especially  on  adding  an  equal  volume  of  alcohol  before 
boiling,  the  leucin  can  be  quantitatively  separated  from  the  tyrosin 
(Habermann  and  Ehbenfeld).  The  1-tyrosin  ethyl  ester  crystallizes  in 
colorless  prisms  which  melt  at  108-109°  C.  Tyrosin  can  be  oxidized  with 
the  formation  of  dark-colored  products  by  various  plant  as  well  as  animal 
oxidases,  so-called  tyrosinases  (see  Chapter  I).  By  the  enzyme  occurring 
in  beet-juice  tyrosin  can  be  converted  into  homogentisic  acid  (Goxxer- 
mann  l).  Tyrosin  is  identified  by  its  crystalline  form  and  by  the  following 
reactions: 

Piria's  Test.  Tyrosin  is  dissolved  in  concentrated  sulphuric  acid  by 
the  aid  of  heat,  by  which  ty rosin-sulphuric  acid  is  formed ;  it  is  allowed  to> 
cool,  diluted  with  water,  neutralized  by  BaC03,  and  filtered.  On  the  addi- 
tion of  a  solution  of  ferric  chloride  the  filtrate  gives  a  beautiful  violet  color. 
This  reaction  is  disturbed  by  the  presence  of  free  mineral  acids  and  by  the 
addition  of  too  much  ferric  chloride. 

Hofmaxx's  Test.  If  some  water  is  poured  on  a  small  quantity  of 
tyrosin  in  a  test-tube  and  a  few  drops  of  Millox's  reagent  added  and  then 
the  mixture  boiled  for  some  time,  the  liquid  becomes  a  beautiful  red  and 
then  yields  a  red  precipitate.  Mercuric  nitrate  may  first  be  added,  then, 
after  this  has  boiled,  nitric  acid  containing  some  nitrous  acid. 

Deniges'  Test,  modified  by  C.  Morner,2  is  performed  as  follows:  To> 
a  few  cubic  centimeters  of  a  solution  consisting  of  1  vol.  formaline,  45  vols. 
water,  and  55  vols,  concentrated  sulphuric  acid  add  a  little  tyrosin  in  sub- 
stance or  in  solution  and  heat  to  boiling.  A  beautiful  permanent  green 
coloration  is  obtained. 

Phenylalanin     (phenvl-a-aminopropionic     acid),  C9HnX02 —  CH.,.C6H5 

CH(NH,) 

COOH 

was  first  found  by  E.  Schulze  and  Barbieri  3  in  etiolated  lupin  sprouts. 

It  is  produced  in  the  acid  cleavage  of  protein  substances.     E.  Fischer 

and  his  collaborators  *  obtained  3.38  per  cent  phenylalanin  from  haemo- 


1  Pfluger's  Arch.,  82. 

2  Deniges,  Compt.  rend.,  130;  C.  Morner,  Zeitschr.  f.  physiol.  Chem.,  37. 
s  Ber.  d.  d.  chem.  Gesellsch.,  14,  and  Zeitschr.  f.  physiol.  Chem.,  12. 

4  See  foot-note  5,  page  66. 


74  THE  PROTEIN  SUBSTANCES. 

globin,  3.0  per  cent  from  horn  substance,  2.5  per  cent  from  ovalbumin  and 
casein,  1.5  per  cent  from  fibroin,  and  0.4  per  cent  from  gelatine. 

The  1-phenylalanin  crystallizes  in  small,  shining  leaves  or  fine  needles 
which  are  rather  difficultly  soluble  in  cold  water  but  readily  soluble  in 
hot  water.  A  5  per  cent  solution  acidified  with  hydrochloric  acid  or  sul- 
phuric acid  is  precipitated  by  phosphotungstic  acid,  while  a  more  dilute 
solution  is  not  precipitated.  On  putrefaction,  phenylalanin  yields  phenyl- 
acetic  acid.  On  heating  with  potassium  dichromate  and  sulphuric  acid 
(25  per  cent)  an  odor  of  phenylacetaldehyde  is  produced  and  benzoic  acid 
is  formed. 

The  separation  and  preparation  of  the  four  amino  acids,  leucin,  aspartic 
acid,  glutamic  acid,  and  tyrosin,  from  a  mixture  of  hydrolytic  decomposi- 
tion products  of  protein  substances  is  performed  essentially  according 
to  the  method  suggested  by  Hlasiwetz  and  Habermann  with  the  modi- 
fications and  changes  proposed  by  other  investigators.  The  isolation  and 
purification  of  the  amino  fatty  acids,  of  phenylalanin,  and  of  a-pyrroli- 
din  carbonic  acid  according  to  FJ.  Fischer  consists  essentially  in  esteriz- 
ing  the  acids  first  with  hydrochloric  acid  and  alcohol,  separating  the 
ester  from  the  hydrochloride  by  means  of  alkali  and  then  fractionally 
distilling  the  ester  under  very  low  pressure,  and  finally  saponifying  the 
different  fractions  by  boiling  with  water  or  by  heating  with  baryta-water. 
It  is  not  within  the  scope  of  this  book  to  give  a  detailed  description  of 
these  methods,  therefore  we  must  refer  for  further  information  to  Hoppe- 
Seyler-Thierfelder's  "Handbuch  der  physiologisch-  und  pathologisch- 
chemischen  Analyse,"  7.  Auflage,  which  also  contains  the  literature  on  the 
subject. 

Cystin  (a-diamino-/?-dithiodi-lactylic  acid), 

C6H12N2SA=CH2-S-S-CH2 

CH(NH2)        CH(NH2) 
COOH  COOH 

was  first  obtained  with  positiveness  as  a  cleavage  product  of  protein  sub- 
stances by  K.  Morner,  and  then  also  by  Embden.  Kulz  *  obtained  it 
also  once  as  a  product  of  tryptic  digestion  of  fibrin.  Morner  obtained 
6.8  per  cent  cystin  from  ox-horn,  13.92  per  cent  from  human  hair,  7.62 
per  cent  from  the  membrane  of  the  hen-egg,  2.53  per  cent  from  seral- 
bumin, 1.51  per  cent  from  serglobulin,  1.17  per  cent  from  fibrinogen,  and 
0.29  per  cent  from  ovalbumin. 

Erlenmeyer,  Jr.,2  has  prepared  cystein  and  cystin  synthetically  from  formyl- 
hippuric  acid  ester  with  monobenzoylserin  ester  and  benzoylthioserin  ester  as 
intermediate  steps. 

1  K.  Morner,  Zeitschr.  f.  physiol.  Chem.,  28  and  34;  Embden,  ibid.,  32;  Kulz, 
Zeitschr.  f.  Biologie,  27. 

2  Ber.  d.  d.  chem.,  Gesellsch.,  36. 


CYSTIN.  75 

Cystin  occurs  in  rare  cases  in  the  urine  or  as  a  calculus,  and  has  also  been 
found  in  ox-kidneys,  in  the  liver  of  the  horse  and  dolphin,  and  as  traces 
in  the  liver  of  a  drunkard.  Abderhalden  '  has  found  cystin  in  the  urine, 
and  also  abundantly  in  the  organs  (spleen)  in  a  case  of  cystin  diathesis. 

The  constitution  of  cystin  has  been  explained  by  Friedmann,2  and 
he  has  also  established  the  relationship  between  cystin  and  taurin.  Cystin 
is  the  disulphide  of  cystein,  which  is  a-amino-/?-thiolactic  acid.  From 
cystein  Friedmann  obtained  cysteinic  acid  (aminosulphopropionic  acid), 

CH2S02OH 
C3H7NS06=CH(NH2),  from  which  taurin  is  produced  by  splitting  off  C02. 
COOH 

Cystin  crystallizes  in  thin,  colorless,  hexagonal  plates.  It  is  not  soluble 
in  water,  alcohol,  ether,  or  acetic  acid,  but  dissolves  in  mineral  acids  and 
oxalic  acid.  It  is  also  soluble  in  alkalies  and  ammonia,  but  not  in  ammo- 
nium carbonate.  Cystin  is  optically  active,  and  indeed  kevorotatnrv. 
Morner  found  it  to  be  (a)D=  —224.3°.  On  heating  with  hydrochloric  acid 
it  can  according  to  Morner  be  changed  into  a  modification  crystallizing 
in  needles  and  with  a  weaker  laevorotatory  power,  and  indeed  it  can  be 
changed  into  a  dextrorotatory  modification.  On  boiling  cystin  with 
caustic  alkali  it  decomposes  and  yields  alkali  sulphide,  which  can  be  de- 
tected by  lead  acetate  or  sodium  nitroprusside.  According  to  Morner,3 
75  per  cent  of  the  total  sulphur  is  separated.  On  treatment  of  cystin 
with  tin  and  hydrochloric  acid  it  develops  only  little  sulphuretted  hydro- 
gen, and  it  is  converted  into  cystein.  On  shaking  a  solution  of  cystin 
in  an  excess  of  sodium  hydrate  with  benzoyl  chloride  a  voluminous  pre- 
cipitate of  benzoyl  cystin  is  obtained  (Baumann  and  Goldmann  4) .  On 
heating  upon  a  platinum  foil  cystin  does  not  melt,  but  ignites  and  burns 
with  a  bluish-green  flame  with  the  generation  of  a  peculiar  sharp  odor. 
When  warmed  with  nitric  acid  it  dissolves  with  decomposition  and  leaves 
on  evaporation  a  reddish-brown  residue,  which  does  not  give  the  murexid 
test.  Cystin  is  gradually  precipitated  from  its  sulphuric  acid  solution 
by  phosphotungstic  acid.  Cystin  forms  crystalline  salts  with  minerals  acids 
and  bases. 

In  the  detection  and  identification  of  cystin  we  make  use  of  the  crystal- 
line form,   the  behavior   on   heating  on  platinum-foil   and   the   sulphur 
reaction   after  boiling  with   alkali.      As  to  its  preparation   from   protein   » 
substances  see  K.  Morner.5    In  regard  to  the  detection  of  cystin  in  the 
urine  see  Chapter  XV. 

1  Zeitschr.  f.  physiol.  Chem.,  38. 

2  Hofmeister's  Beitriige,  3,  1. 

3  Morner,  Zeitschr.  f.  physiol.  Chem.,  34. 

4  Baumann  and  Goldmann,  Zeitschr.  f.  physiol.  Chem.,  12. 
6  Zeitschr.  f.  physiol.  Chem.,  34. 


76  THE  PROTEIN  SUBSTANCES. 

CH2.SH 
Cystein  ( a-amino-/?-thiolactic  acid),  C3H7NS02=CH(NH2),  is  formed  from  cys- 

COOH 
tin  by  reduction  with  tin  and  hydrochloric  acid.  It  is  also  produced  in  the  cleavage 
of  protein  substances,  but  this  is  considered  by  Morner  as  a  secondary  formation, 
while  Embden  considers  it  primary.  Besides  the  /?-cystein  Friedmann  claims 
that  an  «-cystein  of  the  mercapturic  acids  (see  Chapter  XV)  could  also  occur  in  the 
animal  body,  but  his  recent  researches  have  shown  that  this  is  not  warranted.1 
Cystein  can  be  readily  converted  into  cystin. 

Towards  alkalies  and  lead  acetate  it  acts  like  cystin.  With  sodium  nitro- 
prusside  and  alkali  it  gives  a  deep  purple-red  coloration;  with  ferric  chloride  the 
solution  gives  an  indigo -blue  coloration  which  quickly  disappears. 

CH3 
Thiolactic  acid  ( a-thiolactic  acid),  C3H6S02  =  CH.SH,  has  been  found  once  as  a 

COOH 
-cleavage  product  of  ox-horn  by  Baumann   and   Suter.     It  has  been  shown  by 
Friedmann  that  this  acid  is  a  regular  cleavage  product  of  keratin  substances,  and 
that  it  can  also  be  obtained  from  the  proteids.    Frankel  2  obtained  the  acid 
from  haemoglobin. 

'  PIT  NTT 

Taurin  3  (aminoethylsulphonic   acid),  C2H7NS03=pTT2'   ~  2  .„,hasnot 

been  obtained  as  a  cleavage  product  of  protein  substances ;  still  its  origin 
from  proteids  has  been  shown  by  Friedmann  by  the  close  relationship 
that  taurin  bears  to  cystin.  Taurin  is  especially  known  as  a  cleavage 
product  of  taurocholic  acid  and  may  occur  to  a  slight  extent  in  the  intestinal 
contents.  Taurin  has  also  been  found  in  the  lungs  and  kidneys  of  oxen 
and  in  the  blood  and  muscles  of  cold-blooded  animals. 

Taurin  crystallizes  in  colorless,  often  in  large,  shining,  4-6-sided  prisms. 
It  dissolves  in  15-16  parts  of  water  at  ordinary  temperatures,  but  rather 
more  easily  in  warm  water.  It  is  insoluble  in  absolute  alcohol  and  ether; 
in  cold  spirits  of  wine  it  dissolves  slightly,  but  better  when  warm.  Taurin 
yields  acetic  and  sulphurous  acids,  but  no  alkali  sulphides,  on  boiling  with 
strong  caustic  alkali.  The  content  of  sulphur  can  be  determined  as  sul- 
phuric acid  after  fusing  with  saltpeter  and  soda.  Taurin  combines  with 
metallic  oxides.  The  combination  with  mercuric  oxide  is  white,  insoluble, 
and  is  formed  when  a  solution  of  taurin  is  boiled  with  freshly  precipitated 
mercuric  oxide  (J.  Lang  4)-  This  combination  may  be  used  in  detecting 
the  presence  of  taurin.     Taurin  is  not  precipitated  by  metallic  salts. 

The  preparation  of  taurin  from  bile  is  very  simple.     The  bile  is  boiled  a 
few  hours  with  hydrochloric  acid.     The  filtrate  from  the  dyslysin  and 

1  Friedmann,  Hofmeister's  Beitriige,  3  and  4. 

2  Suter,  Zeitschr.  f.   physiol.   Chem.,  20;    Friedmann,   Hofmeister's   Beitrage,  3; 
Frankel,  Sitz.-Ber.  d.  Wien.  Akad.,  112,  II,  b.,  1903. 

3  Taurin  does  not  belong  to  the  cleavage  products  of  the  proteids,  but  for  practical 
reasons  it  will  be  described  in  connection  with  cystin. 

4  See  Maly's  Jahrcsber.,  fi. 


ARGININ.  77 

choloidic  acid  is  concentrated  well  on  the  water-bath  and  filtered  hot  so 
as  to  remove  the  common  salt  and  other  substances  which  have  separated. 
Then  evaporate  to  dryness  and  dissolve  the  residue  in  5  per  cent  hydro- 
chloric acid  and  then  precipitate  with  10  vols.  95  per  cent  alcohol.  The 
crystals  are  readily  purified  by  recrystallization  from  water.  The  alcoholic 
solution  can  l>e  used  for  the  preparation  of  glycocoll.  After  the  evapora- 
tion of  the  alcohol  the  residue  is  dissolved  in  water,  treated  with  a  solution 
of  lead  hydroxide,  filtered,  the  lead  removed  by  ILSand  the  filtrate  strongly 
concentrated.  The  crystals  which  separate  are  dissolved  and  decolorized 
by  animal  charcoal  and  the  solution  then  evaporated  to  crystallization. 

Though  taurin  shows  no  positive  reactions,  it  is  chiefly  identified  by 
its  crystalline  form,  by  its  solubility  in  water  and  insolubility  in  alcohol,  by 
its  combination  with  mercuric  oxide,  by  its  non-precipitability  by  metallic 
salts,  and  above  all  by  its  sulphur  content. 

2.  Diamino  Acids  (hexon  bases). 
Arginin  (guanidine  -«-aminovalerianic  acid), 

(hn)c<nh!ch2 

C6H14N402=  (CH2)2 

CH(NH2) 
COOH 

first  discovered  by  Schulze  and  Steiger  in  etiolated  lupin  and  pumpkin- 
sprouts,  has  later  been  found  in  other  germinating  plants,  in  tubers  and 
roots.  Gulewitsch  has  found  arginin  in  the  ox-spleen.  It  was  first 
found  by  Hedin  as  a  cleavage  product  of  horn  substance,  gelatine,  and 
several  proteids,  and  then  by  Kossel  and  his  pupils  as  a  general  cleav- 
age product  of  protein  substances  as  a  class.  The  greatest  quantity  was 
obtained  from  the  protamins;  but  also  the  histons  and  certain  plant  pro- 
teids (edestin  and  the  proteid  from  pine  seeds)  yield  abundant  arginin. 
Arginin  also  occurs  among  the  products  of  tryptic  digestion  (Kossel 
and  Kutscher  *).  On  boiling  with  baryta- water  arginin  yields  urea  and 
ornithin.  Arginin  has  been  prepared  synthetically  from  ornithin  (a-di- 
amino valerianic  acid)  and  cyanimid  by  Schulze  and  Wintersteix.2 

Arginin  crystallizes  in  rosette-like  tufts,  plates,  or  thin  prisms,  is  readily 
soluble  in  water  and  nearly  insoluble  in  alcohol.  It  forms  crystalline  salts 
or  double  salts  with  several  acids  and  metallic  salts.  Its  acidified  watery 
solution  is  precipitated  by  phosphotungstic  acid.  The  most  important  salts 
are  the  copper-nitrate  (C6H11N402)2.Cu(N03)2  +  3H20  and  the  silver  salts 
C6H14N402-HN03+AgN03  (the  most  readily  soluble)  and  CeH1.,X1( \.AgX03 
+  £H,0  (the  most  difficultly  soluble).  Arginin  is  dextrorotatory,  but  the 
arginin  obtained  by  Kutscher  in  the  tryptic  digestion  of  fibrin  was  inactive. 

1  Schulze  and  Steiger,  Zeitschr.  f.  physiol.  Chem.,  11;  Gulewitsch,  ibid.,  30;  Hedin, 
{bid.,  20  and  21;  Kossel  and  Kutscher,  ibid.,  22,  25,  26. 

2  Ber.  d.  d.  chem.  Gesellsch.,  32,  and  Zeitschr.  f.  physiol.  Chem.,  34. 


78  THE  PROTEIN  SUBSTANCES. 

CH2.(NH2) 
(hfx  \ 
Ornithin   ( a-diaminovalerianic  acid),    C5H12N202=   PHfNH  y  is  not  a  primary- 

COOH 
cleavage  product  of  proteids,  but  is  formed  from  arginin  on  boiling  with  baryta- 
water.  Jaffe,1  who  first  discovered  this  body,  obtained  it  as  a  cleavage  product 
from  ornithuric  acid,  which  is  found  in  the  urine  of  hens  fed  with  benzoic  acid. 
The  ornithin  which  E.  Fischer2  prepared  synthetically  yields,  as  shown  by 
Ellinger,3  putrescin  (tetramethylendiamine).  C4H8(NH2)2,  on  putrefaction. 

Ornithin  is  a  non-crystalline  substance  which  dissolves  in  water,  giving  an 
alkaline  reaction  and  yields  several  crystalline  salts.  It  is  precipitated  by 
phosphotungstic  acid  and  several  metallic  salts,  but  not  by  silver  nitrate  and 
baryta- water  (differing  from  arginin).  Ornithin  hydrochloride  is  dextrorotatory; 
the  synthetically  prepared  is  inactive.  On  shaking  ornithin  with  benzoyl  chloride 
and  caustic  soda  it  is  converted  into  dibenzoyl  ornithin  (ornithuric  acid). 

Diaminoacetic  acid,  C2H6N202  =  CH(NH2)2COOH,  was  obtained  by  Drechsel4 
as  a  cleavage  product  of  casein  by  boiling  with  tin  and  hydrochloric  acid.  It 
crystallizes  in  prisms  and  gives  a  monobenzoyl  compound  which  is  not  very  soluble 
in  cold  water  and  nearly  insoluble  in  alcohol  and  can  be  used  in  the  isolation  of 
the  acid. 

CH2(NH2) 
Lysin  (a-s-diaminocaproic  acid),  C6H14N202=  p^iVr  y  was  first  obtained 

COOH 

by  Drechsel  as  a  cleavage  product  of  casein.  Later  he  and  his  pupils, 
as  well  as  Kossel  and  others,  found  it  among  the  cleavage  products  of 
various  proteids.  It  has  not  been  detected  in  certain  vegetable  pro- 
teids such  as  zein  and  gluten-proteid.  E.  Schtjlze  found  lysin  in  ger- 
minating plants  of  the  lupinus  luteus,  and  Winterstein  5  found  it  in  ripe 
cheese. 

Lysin  has  been  synthetically  prepared  by  E.  Fischer  and  Weigert.6 
This  lysin  was  inactive,  while  that  prepared  from  proteid  is  always  optic- 
ally active  and  dextrorotatory.  On  heating  with  barium  hydrate  it  is 
converted  into  the  inactive  modification.  According  to  Ellinger7  lysin 
yields  cadaverin  (pentamethylendiamine) ,  C5H10(XH2)2,  on  putrefaction. 

Lysin  is  readily  soluble  in  water  but  is  not  crystalline.  The  aqueous 
solution  is  precipitated  by  phosphotungstic  acid  but  not  by  silver  nitrate 

1  Ber.  d.  d.  chem.  Gesellsch.,  10  and  11. 

2  Ibid.,  34. 

3  Zeitschr.  f.  physiol.  Chem.,  29. 

4  Ber.  d.  siichs.  Ges.  d.  Wissensch. ,  44. 

s  Drechsel,  Arch.  f.  (Anat.  u.)  Physiol.,  1891,  and  Ber.  d.  d.  chem. Gesellsch.,  25;  Sieg- 
fried, Arch.  f.  (Anat.  u.)  Physiol.,  1891,  and  Ber.  d.  d.  chem.  Gesellsch.,  24;  Hedin, 
Zeitschr.  f.  physiol.  Chem.,  21;  Kossel,  ibid.,  25;  Kossel  and  Mathews,  ibid.,  25; 
Kossel  and  Kutscher,  ibid.,  31;  Kutscher,  ibid.,  29;  Schulze,  ibid.,  28;  Winterstein,. 
cited  in  Schulze  and  Winterstein,  Ergebnisse  der  Physiologie  I,  Abt.  I,  1902. 

8  Ber.  d.  d.  chem.  Gesellsch.,  35. 

7  Zeitschr.  f.  physiol.  Chem.,  29. 


1I1ST1DIN.  79 

and  baryta-water  (differing  from  arginin  and  histidin).  It  gives  two 
hydrochlorides  with  hydrochloric  acid  and  with  platinum  chloride  a  chlo- 
roplatinate  which  is  precipitable  by  alcohol  and  has  the  composition 
C6H14X202.H2PtCl6+C2H5OH.  It  gives  two  silver  salts  with  AgNO,;  one 
has  the  formula  AgX03  +  CuHuX./)2  and  the  other  AgX03  +  C6HuXXULV  >:j. 
With  benzoyl  chloride  and  alkali  lysin  forms  an  acid,  lyswric  acid, 
C6H12(C7H50)2X202  (Drechsel),  which  is  homologous  with  ornithuric  acid 
and  whose  difficultly  soluble  acid  barium  salt  may  be  used  in  the 
ration  of  lysin.1  The  rather  insoluble  picrate,  which  Ls  precipitated  from 
a  not  too  dilute  solution  of  the  hydrochloride  by  sodium  picrate,  may  be 
used  in  the  detection  of  lysin. 

Lysatin  or  lysatinin.  The  formula  of  this  substance  is  either  C,H13X302  or 
CeH1}N30+  H20.  In  the  first  case  this  base  is  a  homologue  of  creatine,  C4H9X302, 
and  in  the  other  case  a  homologue  of  creatinine,  C4H7X30,  and  this  is  the  reason 
why  this  body  is  called  lysatin  as  well  as  lysatinin.  It  is  still  a  question  whether 
lysatin  is  a  chemical  individual  or,  as  Hedin  suggests,  only  a  mixture  of  lysin 
and  arginin.2 

Histidin,  C6H9N302,  is,  according  to  the  investigations  of  S.  Frankel,3 
not  a  diamino  acid,  but  more  probably  aminomethyldehydropyrimidin  car- 
bonic acid.  As  it  is  always  obtained  with  the  diamino  acids  it  is  called 
a  hexon  base,  hence  it  will  be  treated  here  with  the  diamino  acids.  Histidin 
was  first  discovered  by  Kossel  in  the  cleavage  products  of  sturin.  It 
was  then  found  by  Hedin  in  the  cleavage  products  of  proteids  by  acid 
hydrolysis  and  by  Kutscher  among  the  products  of  tryptic  digestion,  and 
finally  also  as  a  cleavage  product  of  different  protein  substances.  It  also 
occurs  in  germinating  plants  (E.  Schulze).4 

Histidin  crystallizes  in  colorless  needles  and  plates  and  is  readily  soluble 
in  water,  but  less  soluble  in  alcohol,  and  has  an  alkaline  reaction.  It 
is  precipitated  by  phosphotungstic  acid,  but  this  precipitate  is  soluble 
in  an  excess  of  the  precipitant  (Frankel).  With  silver  nitrate  alone  the 
aqueous  solution  is  not  precipitated;  on  the  careful  addition  of  ammonia 
or  baryta-water  an  amorphous  precipitate,  which  is  readily  soluble  in 
an  excess  of  ammonia,  is  obtained.  Histidin  can  be  precipitated  by  mer- 
curic chloride,  or,  still  better,  by  the  sulphate  acidified  with  sulphuric  acid, 
and  can  in  this  wTay  be  separated  from  the  diamino  acids  as  well  as  from 
the  monamino  acids  (Kossel  and  Patten).  The  hydrochloride  crystal- 
lizes in  beautiful  plates  (Bauer),  dissolves  rather  readily  in  water,  but  is 

1  Drechsel,  Ber.  d.  d.  chem.  Gesellsch.,  28;  see  also  C.  Willdenow,  Zeitschr.  f. 
physiol.  Chem.,  25. 

7  Hedin,  Zeitschr.  f.  physiol.  Chem.,  21;  Siegfried,  ibid.,  35. 

sSitz.-Ber.  d.  Wicn.  Akad.,  112,  lib,  1903. 

*  Kossel,  Zeitschr.  f.  physiol.  Chem.,  22;  Hedin,  ibid.,  Kutscher,  ibid.,  25;  Wetzel, 
ibid.,  26;  Lawrow,  ibid.,  2S,  and  Ber.  d.  d.  chem.  Gesellsch.,  34;  Kossel  and  Kutscher, 
Zeitschr.  f.  physiol.  Chem.,  31;  Hart,  ibid.,  33;  Schulze,  ibid.,  24  and  2S. 


SO  THE  PROTEIN  SUBSTANCES. 

insoluble  in  alcohol  and  ether.  Histidin  is  laevorotatory,  while  its  solu- 
tion in  hydrochloric  acid  is  dextrorotatory.  On  warming  it  gives  the 
biuret  test  (Herzog  1),  and  it  also  gives  Weidel's  reaction  if  performed 
as  suggested  by  Fischer  (see  Xanthine,  Chapter  V)  (Frankel). 

In  the  preparation  of  the  above  bases  we  can  first  precipitate  all  the 
bases  by  phosphotungstic  acid,  when  the  monamino  acids  remain  in  solu- 
tion. The  precipitate  is  decomposed  in  boiling  water  by  barium  hydrate 
and  the  bases  obtained  as  silver  compounds  from  this  filtrate.  In  regard 
to  further  details  we  must  refer  to  the  cited  works  of  Drechsel  and  Hedin. 
Kossel  and  Kutscher  2  have  suggested  a  method  of  separating  histidin 
and  arginin  as  silver  compounds  from  lysin,  and  Kossel  and  Patten 
have  proposed  a  method  of  separating  histidin  from  arginin  by  means  of 
mercuric  sulphate. 

We  give  below  a  tabulation  of  the  amounts  of  the  three  hexon  bases 
found  in  certain  protein  substances  (in  weight  per  cent) : 

Arginin  Lysin  Histidin 

Sturin3 58.2  12.0  12.9 

Other  protamins  3 62.5 — 84.3  0.0  0.0 

Histons3 14.36—15.52  7.7—8.3  1.21—2.34 

Casein4 '. 4.70^.84  1.92—5.80  2.53—2.59 

Syntonin  (from  meat)  4 5 .  06  3 .  26  2 .  66 

Heterosyntonose 4 8.53  3.08—7.03  0.37—1.12 

Protosyntonose  4 4 .  55  3 .  08  3 .  35 

Edestin5 11.0—14.07  1.3  1.17 

Proteid  from  conif erse  seeds  5 10 . 9 — 1 1.3  0 .  25 — 0 .79  0 .  62 — 0 .  78 

Gluten  casein  3 4.4  2.15  1.16 

Gluten  proteins 3 2.75—3.13  0.0  0.43—1.53 

Gelatine  3  and  * 7.62—9.3  2.49—6.0  0.40 

Elastin 6 , 0.3  +  0.027 

3.  Pyrrol  and  Indol  Derivatives. 

CH2 — CHa 

I         I 
a-Pyrrolidin  carbonic  acid,  C5H9N02=CH2  CH.COOH,  was  prepared 


NH 

by  E.  Fischer  as  a  cleavage  product  from  casein  (3.2  per  cent)  and 
tDvalbumin  (1.55  per  cent)  and  by  him  and  his  collaborators  in  the  tryptic 
•digestion  of  casein,  and  as  a  cleavage  product  of  hsemoglobin  (1.46  per 

1  Kossel  and    Patten,  Zeitschr.  f.  physiol.  Chem.,  38;    Bauer,  ibid.,  22;    Herzog, 
ibid.,  87. 

2  Zeitschr.  f.  physiol.  Chem.,  31;  Kossel  and  Patten,  1.  c. 

3  Kossel  and  Kutscher,  Zeitschr.  f .  physiol   Chem. ,  31. 

4  Hart,  ibid.,  33. 

5  Schulze  and  Winterstein,  ibid.,  33;   see  also  Kossel,  Ber.  d.  d.  chem.  Gesellsch., 
34,  3236. 

8  Kossel  and  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  25,  and  Richards   and  Gies, 
Amer.  Journ.  of  Physiol.,  7. 


SKATOLAMINOACETIC  ACID.  81 

cent),  gelatine  (5.2  per  cent),  horn  substance  (3.00  per  cent),  and  from 
silk  fibroin.1  The  acid  thus  obtained  was  generally  the  hcvorotatory 
modification. 

This  acid  is  readily  soluble  in  water  and  alcohol  and  crystallizes  in  flat 
needles  which  melt  at  203-200°  C.  with  an  odor  of  pyrrolidine.  The  solu- 
tion acidified  with  sulphuric  acid  is  precipitated  by  phosphotungstic  acid. 
In  the  detection  of  this  acid  we  make  use  of  the  copper  salt  and  the  an- 
hydride of  the  phenylisocyanate  compound.2  The  inactive  acid  and  its 
compounds  show  somewhat  varying  properties.  In  regard  to  the  prepa- 
ration of  this  acid  we  refer  to  p.  74. 

In  the  hydrolysis  of  gelatine  and  casein  E.  Fischer  3  obtained  an  amino 
acid  having  the  formula  C5H9N03,  which  on  reduction  yielded  a-pyrrolidin 
carbonic  acid,  and  which  according  to  Fischer  is  an  oxypyrrohdin-a- 
carbonic  acid. 

Skatolaminoacetic  acid  (tryptophan,  proteinochromogen),  CnH12N202  = 
C.CH3 

/\ 
C6H4       C.CH(NH2)COOH,  is  one  of   the  cleavage  products  of  the  pro- 
NX 
NH 

teids  formed  in  tryptic  digestion  and  other  deep  decompositions  of  the 
proteids,  such  as  putrefaction,  cleavage  with  baryta-water  or  sulphuric 
acid,  but  not  in  peptic  digestion.  It  gives  a  reddish-violet  product  with 
chlorine  or  bromine  which  is  called  proteinochrome.  Nencki  4  considered 
tryptophan,  which  name  is  generally  given  to  this  acid,  as  the  mother- 
;substance  of  various  animal  pigments. 

The  preparation  of  tryptophan  was  for  a  long  time  impossible  until 
Hopkins  and  Cole  5  prepared  a  crystalline  substance  which  they  con- 
sidered pure  tryptophan.  This  substance,  skatolaminoacetic  acid,  crystal- 
lizes in  shining  plates,  which  are  readily  soluble  in  hot  water,  less  soluble  in 
cold  water  and  in  alcohol.  On  heating  sufficiently,  it  yields  indol  and 
skatol.  It  gives  the  Adamkiewicz-Hopkins  reaction  and  a  rose-red  colora- 
tion on  the  addition  of  bromine  water  (tryptophan  reaction).  If  a  pine 
stick  moistened  with  hydrochloric  acid  and  then  washed  off  be  introduced 
into  a  concentrated  tryptophan  solution,  it  becomes  purple-colored  on  drying 

1  E.  Fischer,  Zeitschr.  f.  physiol.  Chem.,  33  and  35;  also  Fischer  and  Abderhalden, 
ibid.,  40.     See  also  foot-note  5,  page  66. 

2  In  regard  to  the  preparation  of  the  phenylisocyanate  compounds  of  the  amino 
acids,  see  Paal,  Ber.  d.  d.  chem.  Gesellsch.,  27;  Mouneyrat,  ibid.,  33,  and  Hoppe- 
Seyler-Thierf elder 's  Handbuch,  7.  Aufl. 

3  Ber.  d.  d.  chem.  Gesellsch.,  3o  and  36. 

4  In  regard  to  trytophan,  see  Stadelmann,  Zeitschr.  f.  Biologie,  26;  Neumeister, 
ibid.,  26;  Nencki,  Ber.  d.  d.  chem.  Gesellsch.,  28;  Beitler,  ibid.,  31;  Kurajeff,  Zeitschr. 
f.  physiol.  Chem.,  26;  Klug,  Pfluger's  Arch.,  86. 

6  Journ.  of  Physiol.,  2". 


S2  THE  PROTEIN  SUBSTANCES. 

(pyrrol  reaction).  Tryptophan,  as  Hopkins  and  Cole  l  showed  later,  yields 
skatolacetic  acid  on  anaerobic  putrefaction,  and  skatolcarbonic  acid,  skatol, 
and  indol  on  aerobic  putrefaction. 

In  regard  to  the  '  somewhat  complicated  method  of  preparation  we 
must  refer  to  the  original  work  of  Hopkins  and  Cole. 

Skatosin,  C10H16N2O2,  is  a  base  first  obtained  by  Baum  in  the  pancreas  auto- 
digestion  and  later  studied  by  Swain.  It  develops  an  indol-  or  skatol-like  odor 
on  fusing  with  potassium  hydrate.  Langstein  2  obtained  a  substance,  which  is. 
perhaps  identical  with  skatosin,  in  the  very  lengthy  peptic  digestion  of  blood 
proteid. 

The  putrefactive  products  of  the  proteids  will  be  in  part  treated  in 
Chapter  IX  (intestinal  putrefaction)  and  in  part  in  Chapter  XV  (putre- 
factive products  in  the  urine). 

1  Journ.  of  Physiol.,  29;  see  also  Ellinger  and  Gentzen,  Hofmeister's  Beitrage,  4. 

2  Baum,  Hofmeister's  Beitrage,  3;  Swain,  ibid.;  Langstein,  see  Hofmeister,  Uber 
Bau  und  Gruppierung  der  Eiweisskorper,  in  Ergebnisse  der  Physiologie,  I,  Abt.  I,  1902. 


CHAPTER  III. 
THE  CARBOHYDRATES. 

We  designate  with  this  name  bodies  which  are  especially  abundant 
in  the  plant  kingdom.  As  the  protein  bodies  form  the  chief  portion  of 
the  solids  in  animal  tissues,  so  the  carbohydrates  form  the  chief  portion 
of  the  dry  substance  of  the  plant  structure.  They  occur  in  the  animal 
kingdom  only  in  proportionately  small  quantities  either  free  or  in  com- 
bination with  more  complex  molecules,  forming  compound  proteids. 
Carbohydrates  are  of  extraordinarily  great  importance  as  food  for  both 
man   and   animals. 

The  carbohydrates  contain  only  carbon,  hydrogen,  and  oxygen.  The 
last  two  elempnts  occur,  as  a  rule,  in  the  same  proportion  as  they  do  in 
water,  namely,  2:1,  and  tins  is  the  reason  why  the  name  carbohydrates 
has  been  given  to  them.  This  name  is  not  quite  pertinent,  if  strictly  con- 
sidered because  even  though  we  have  bodies,  such  as  acetic  acid  and 
'lactic  acid,  which  are  not  carbohydrates  and  still  have  their  oxygen  and 
hydrogen  in  the  proportion  as  in  water,  nevertheless  we  also  have  a  sugar 
(rhamnose,  C6H1205)  which  has  these  two  elements  in  another  proportion. 
Heretofore  it  was  thought  possible  to  characterize  as  carbohydrates  those 
bodies  which  contained  6  atoms  of  carbon,  or  a  multiple,  in  the  molecule, 
but  this  is  not  considered  valid  at  the  present  time.  We  have  true  carbo- 
hydrates containing  less  than  6  and  also  those  containing  7,  8,  and  9  car- 
bon atoms  in  the  molecule.  The  carbohydrates  have  no  properties  or 
characteristics  in  general  which  differentiate  them  from  other  bodies; 
on  the  contrary,  the  various  carbohydrates  are  in  many  cases  very  different  / 
in  their  external  properties.  Under  these  circumstances  it  is  very  difficult 
\to  give  a  positive  definition  for  the  carbohydrates. 

From  a  chemical  standpoint  we  can  say  that  all  carbohydrates  are 
aldehyde  or  ketone  derivatives  of  polyhydric  alcohols.  The  simplest 
carbohydrates,  the  simple  sugars  or  monosaccharides,  are  either  aldehyde 
or  ketone  derivatives  of  such  alcohols,  and  the  more  complex  carbohydrates 
seem  to  be  derived  from  these  by  the  formation  of  anhydrides,  It  is  a 
fact  that  the  more  complex  carbohydrates  yield  two  or  even  more  molecules 
of  the  simple  sugars  when  made  to  undergo  hydrolytic  splitting. 

The  carbohydrates  are  generally  divided  into  three  chief  groups,  namely, 
monosuccharldt  s,  disaccharidt  s,  and  polysaccharides. 

83 


84  THE  CARBOHYDRATES. 

Our  knowledge  of  the  carbohydrates  and  their  structural  relationships- 
has  recently  been  very  much  extended  by  the  pioneering  investigations 
of  Kiliani  *  and  especially  those  of  E.  Fischer.2 

As  the  carbohydrates  occur  chiefly  in  the  plant  kingdom  it  is  naturally 
not  the  place  here  to  give  a  complete  discussion  of  the  numerous  carbo- 
hydrates known  up  to  the  present  time.  According  to  the  plan  of  this 
work  it  is  only  possible  to  give  a  short  review  of  those  carbohydrates  which 
occur  in  the  animal  kingdom  or  are  of  special  importance  as  food  for  man 
and  animals. 


Monosaccharides. 

All  varieties  of  sugars,  the  monosaccharides  as  well  as  disaccharides, 
are  characterized  by  the  termination  "ose/'  to  which  a  root  is  added 
signifying  their  origin  or  other  relations.  According  to  the  number  of 
carbon  atoms,  or  more  correctly  oxygen  atoms;  contained  in  the  molecule 
the  monosaccharides  are  divided  into  trioses,  tetroses,  pentoses,  hexoses, 
heptoses,  and  so  on. 

All  monosaccharides  are  either  aldehydes  or  ketones  of  polyhydric 
alcohols.  The  first  are  termed  aldoses  and  the  other  ketoses.  Ordinary 
dextrose  is  an  aldose,  while  ordinary  fruit  sugar  (lsevulose)  is  a  ketose.  The 
difference  may  be  shown  by  the  structural  formula  of  these  two  varieties  of 
sugar.: 

Dextrose  =  CH,(OH)  .CH(OH)  .CH(OH)  .CH(OH)  .CH(OH)  .CHO ; 
La3vulose  =  CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH). 

A  difference  is  also  observed  on  oxidation.  The  aldoses  can  be  con- 
verted into  oxyacids  having  the  same  quantity  of  carbon,  while  the  ketoses 
yield  acids  having  less  carbon.  On  mild  oxidation  the  aldoses  yield  mono- 
basic oxyacids  and  dibasic  acids  on  more  energetic  oxidation.  Thus 
ordinary  dextrose  yields  gluconic  acid  in  the  first  case  and  saccharic  acid  in 
the  second. 

Gluconic  acid  =CH2(OH).[CH(OH)]4.COOH; 
Saccharic  acid  =  COOH.[CH(OH)]4.COOH. 

The  monobasic  oxyacids  are  of  the  greatest  importance  in  the  artificial  forma- 
tion of  the  monosaccharides.     These  acids,  as  lactones,  can  be  converted  into 

1  Ber.  d.  deutsch.  chem.  Gesellsch.,  18,  19,  and  20. 

2  See  E.  Fischer's  lecture:  "Synthesen  in  der  Zuckergruppe,"  Ber.  d.  deutsch. 
chem.  Gesellsch.,  23,  2114.  An  excellent  work  on  carbohydrates  is  Tollen's  "Kurzes 
Handbuch  der  Kohlehydrate,"  Breslau,  2,  1895,  and  1,  2.  Auflage,  1898,  which  gives 
a  complete  review  of  the  literature. 


MONOSACCHARIDES.  85- 

their  respective  aldehydes  (corresponding  to  the  sugars)  by  the  action  of  nascent 
hydrogen.  On  the  other  hand,  they  may  be  transformed  into  stereoisomeric 
acids  on  heating  with  quinoline,  pyridine,  etc.,  and  the  stereoisomeric  sugars  may 
be  obtained  from  these  by  reduction. 

Numerous  isomers  occur  among  the  monosaccharides,  and  especially  in  the 
hexose  group.  In  certain  cases,  as  for  instance  in  glucose  and  laevulose,  we  are 
dealing  with  a  different  constitution  (aldoses  and  ketoses),  but  in  most  cases 
we  have  stereoisomerism  due  to  the  presence  of  asymmetric  carbon  atoms. 

The  monosaccharides  arc  converted  into  the  corresponding  polyhydric 
alcohols  by  nascent  hydrogen.  Thus  arabinose,  which  is  a  pentose, 
Cyi^O-,  is  transformed  into  the  pentatomic  alcohol,  arabite,  C5H1205. 
T1kv  three  hexoses,  dextrose,  L/Evulose,  and  galactose,  CqII^O,,,  are 
transformed  into  the  corresponding  three  hexites,  sorbite,  maxxite,  and 
dulctte,  CJI^Og.  In  these  reductions  a  second  isomeric  alcohol  is  also 
obtained;  in  the  reduction  of  laevulose  we  obtain  besides  mannite  also 
sorbite.  Inversely,  the  corresponding  sugars  may  be  preparedfrom 
polyhydric   alcohols  by  careful   oxidation. 

Similar  to  the  ordinary  aldehydes  and  ketones  the  sugars  may  be  made  to 
take  up  hydrocyanic  acid.  Cyanhydrines  are  thus  formed.  These  addition 
products  are  of  special  interest  in  that  they  make  the  artificial  preparation  possi- 
ble of  sugars  rich  in  carbon  from  sugars  poor  in  carbon. 

As  an  example,  if  we  start  from  dextrose  we  obtain  glucocyanhydrin  on  the 
addition  of  hydrocyanic  acid: 

CH2.(OH).[CH(OH)]4.COH+HCN  =  CH2(OH).[CH(OH)]4.CH(OH).CN. 

On  the  saponification  of  glucocyanhydrin  the  corresponding  oxyacid  is  formed: 

CH2(OH)  .[CH(OH)]4.CH(OH)  .CN+  2H20 

=  CH2(OH).[CH(OH)]4.CH(OH).COOH+NH3. 

By  the  action  of  nascent  hydrogen  on  the  lactone  of  this  acid  we  obtain  gluco- 
heptose,  C7H1407. 

The  monosaccharides  give  the  corresponding  oximes  with  hydro xylamine; 
thus  glucose  yields  glucosoxime,  CH2(OH).[CH(OH)]4.CH  :  N.OH.  These  com- 
binations are  of  importance  on  account  of  the  fact,  as  found  by  Wohl,1  that 
they  are  the  starting-point  in  the  building  up  of  varieties  of  sugars,  namely,  the 
preparation  of  sugars  poor  in  carbon  from  those  rich  in  carbon. 

The  monosaccharides  are  strong  reducing  bodies,  similar  to  the  alde- 
hydes. They  reduce  metallic  silver  from  ammoniaeal  silver  solutions,  and 
also  several  metallic  oxides,  such  as  copper,  bismuth,  and  mercury  oxides, 
on  warming  their  alkaline  solutions.  TKis  property  is  of  the  greatest, 
importance  in  their  detection  and  quantitative  estimation. 

With  phenylhydrazine  or  substituted  phenylhydrazines  the  sugars  first 
yield  hydrazoncs  with  the  elimination  of  water,  and  then  on  the  further 
action  of  hydrazine  on  warming  in  an  acetic  acid  solution  we  obtain  osazones. 

1  Ber.  d.  deutsch.  chem.  Gesellsch.,  26. 


86  THE  CARBOHYDRATES. 

The  reaction  takes  place  as  follows: 

(a)  CH2(OH).[CH(OH)]3.CH(OH).CHO+  H2N.NH.C6H5 

=  CH,(OH).LCH(OH)l3.CH(OH)CH  :  N.NH.C6H5+  H20. 

Phenylglucoshydrazone. 

(b)  CH2(OH)[CH(OH)]3.CH(OH).CH  :  N.NH.C6H5+H2N.NH.C6H5 

=  CH2(OH).[CH(OH)]3.C  .  CH  :  N.NH.C6H5 

N.NH.CaHB+H20+H2. 

Phenylglucosazone. 

The  hydrogen  is  not  evolved,  but  acts  on  a  second  molecule  of  phenylhy- 
drazine  and  splits  it  into  aniline  and  ammonia: 

H2N.NH.C6H5+  H2  =  H2N.C6H5+ NH3. 

The  osazones  are  generally  yellow  crystalline  combinations  which  differ 
from  each  other  in  melting-point,  solubility,  and  optical  properties,  and 
hence  have  received  great  importance  in  the  characterization  of  certain 
sugars.  They  have  also  become  of  extraordinarily  great  interest  in 
the  study  of  the  carbohydrates  for  other  reasons.  Thus  they  are  a  very 
good  means  of  precipitating  sugars  from  solution  in  which  they  occur  mixed 
with  other  bodies,  and  they  are  of  the  greatest  importance  in  the  artificial 
preparation  of  sugars.  On  cleavage,  by  the  short  action  of  gentle  heat  and 
fuming  hydrochloric  acid  (for  disaccharides  still  better  with  benzaldehyde)  - 
the  osazones  yield  so-called  osones,  which  on  reduction  yield  glucoses  and 
more  often  ketoses. 

We  can  also  pass  from  the  osazones  to  the  corresponding  sugars 
(ketoses)  in  other  ways,  namely,  by  direct  reduction  of  the  osazones  with 
acetic  acid  and  zinc  dust.  The  corresponding  osamine  is  first  formed 
(from  phenylglucosazone  we  obtain  isoglucosamine),  which  on  treatment 
with  nitrous  acid  yie"  ds  the  sugar  (in  this  case  lsevulose). 

The  sugars  can  be  prepared  from  the  hydrazones  by  decomposition 
with  benzaldehyde  (Herzfeld)  or  with  formaldehyde  (Ruff  and  Ollen- 
dorff 2) .  This  latter  method  is  especially  applicable  if  substituted  hydra- 
zines, especially  benzylphenylhydrazine,  is  used. 

With  ammonia  the  glucoses  may  form  compounds  which  have  been 
considered  as  osamines  by  Lobry  de  Bruyn,  but  to  differentiate  them  from 
the  true  osamines  have  been  called  osimines  by  E.  Fischer.3  The  corre- 
sponding osaminic  acid  can  be  obtained  from  such  an  osimine  by  the  action 
of  ammonia  and  hydrocyanic  acid,  and  from  the  hydrochloric-acid  lactone 
of  this  acid  the  osamine  is  obtained  by  reduction  with  sodium  amalgam. 
In  this  manner  E.  Fischer  and  Leuchs  artificially  prepared  d-glucosa- 
mine,  which  occurs  in  the  animal  kingdom  and  is  an  isomer  of  the  above- 


1  E.  Fischer  and  Armstrong,  Ber.  d.  d.  chem.  Gesellsch.,  35. 

2  Herzfeld,  ibid.,  28;   Ruff  and  Ollendorff,  ibid.,  32. 
8  Lobry  de  Bruyn,  ibid.,  28;   E.  Fischer,  ibid.,  35. 


MONOS.  iCCHA  RIDES.  '     87 

mentioned  isoglucosamine,  by  starting  from  d-arabinose,  then  obtaining 
d-arabinosiniinc,  then  d-glueosaminie  acid,  and  finally  the  glucosamine 
from  the  Lactone  <»!'  this  acid.  They  l  also  have  prepared  1-glucosamine 
from  1-arabinose  in  a  similar  manner. 

By  the  action  of  hydrochloric  acid  upon  alcoholic  sugar  solutions  E. 
Fischer  and  his  pupils  have  obtained  ether-like  compounds  which  have 
been  called  glucosides.  Compounds  with  aromatic  groups  similar  to  the 
glucosides  occur  widely  distributed  in  the  vegetable  kingdom.  The  more 
complex  carbohydrates  may  be  considered,  according  to  Fischer,  as 
glucosides  of  the  sugars.  Thus  maltose,  for  example,  is  the  glucoside  and 
lactose  the  galactoside  of  dextrose. 

By  the  action  of  alkalies,  even  in  small  amounts,  as  also  of  alkaline  earths 
and  lead  hydroxide,  a  reciprocal  transformation  of  the  sugars,  such  as 
dextrose,  lsevulose,  and  mannose,  may  take  place  (Lobry  de  Bruyn  and 

AiBERDA    VAX   ElCENSTEIN2). 

Four  other  sugars,  among  them  two  kctoses,  are  produced  by  the  action  of 
potash  or  soda  on  each  of  the  three  sugars,  dextrose,  lawulose,  and  galactose. 
For  example,  from  dextrose  two  ketoses,  lsevulose  and  pseudola:vulose,  are  pro- 
duced, also  mannose  and  a  non-fermentable  sugar,  glutose.  From  galactose 
are  formed  talose  and  galtose,  besides  two  ketoses,  tagatose  and  pseudotagatose. 

Thp  transform fiti on  of  the  different  varieties  of  sugar  into  each  other 
also  occurs  in  the  animal  body.  Neuberg  and  Mayer  3  have  shown  by 
experiments  on  rabbits  the  partial  transformation  of  various  mannoses  into 
the  corresponding  glucoses. 

The  monosaccharides  are  colorless  and  odorless  bodies,  neutral  in  reac- 
tion, with  a  sweet  taste,  readily  soluble  in  water,  generally  soluble  with 
difficulty  in  absolute  alcohol,  and  insoluble  in  ether.  Some  of  them 
crystallize  well  in  the  pure  state.  They  are  optically  active,  some  la?vo- 
rotatory  and  others  dextrorotatory ;  but  there  are  also  optically  inactive 
modifications  (racemic),  which  are  formed  from  two  optically  opposed  com- 
ponents. 

We  designate  the  optical  activity  of  the  carbohydrates  with  the  letter  1- 
for  kevogyrate,  d-  for  dextrogyrate,  and  i-  for  inactive.  These  are  only 
partly  useful.  Thus  dextrorotatory  glucose  is  designated  d-glucose,  kevo- 
rotatory  1-glucose,  and  the  inactive  i-glucose.  Emil  Fischer  has  used  these 
signs  in  another  sense.  He  designates  by  these  signs  the  mutual  relationship 
of  the  various  kinds  of  sugars  instead  of  their  optical  activity.  For  exam- 
ple, he  does  not  designate  the  laevorotatory  kevulose  1-kevulose,  but  d-lsevu- 
lose,  showing  its  close  relation  to  dextrorotatory  d-glucose.      This  designa- 

1  Ber.  d.  d.  chem.  Gesellsch.,  36  and  35,  3787. 

1 1bid.,  28,  3078;  Bull.  soc.  chim.  de  Paris  (3),  15j  Chem.  Centralbl.,  1896,  2,  and 
1897,  2. 

3  Zeitschr.  f.  physiol.  Chem.,  3". 


88  THE  CARBOHYDRATES. 

tion  is  generally  accepted,  and  the  above-mentioned  signs  only  show  the- 
optical  properties  in  a  few  cases. 

Specific  rotation  means  the  rotation  in  degrees  produced*  by  1  gm.  substance 
dissolved  in  1  cc.  liquid  placed  in  a  tube  1  dcm.  long.  The  reading  is  ordinarily 
made  at  20°  C.  and  with  a  homogeneous  sodium  light.  The  specific  rotation  with 
this  light  is  represented  by  (a)D,  and  is  expressed  by  the  following  formula: 

(a)D  =  ± — r,  in  which  a  represents  the  reading  of  degrees,  1  the  length  of  the 

tube  in  decimetres,  and  p  the  werht  of  substance  in  1  cc.  of  the  liquid.      In- 
versely the  per  cent  P  of  substance  can  be  calculated,  when  the  specific  rotation 

is  known,  by  the  formula  P= — =-,  in  which  s  represents  the  known  specific 

rotation.  ■• 

A  freshly  prepared  sugar  solution  often  shows  another  rotation  from  that 
after  it  has  been  allowed  to  stand  for  some  time.  If  the  rotation  gradually 
diminishes,  this  is  called  birotation,  while  a  gradual  increase  in  the  rotation  is 
called  half -rotation. 

Many  monosaccharides,  but  not  all,  ferment  with  yeast,  and  it  has  been 
shown  that  only  those  varieties  of  sugar  containing  3,  6,  or  9  atoms  of 
carbon  in  the  molecule  are  fermentable  with  yeast.  We  must  state,  how- 
ever, that  the  power  of  fermentation  with  pure  yeast  has  been  shown 
only  for  the  hexose  group,  and  in  fact  all  of  the  hexoses  do  not  ferment. 
The  restriction  of  fermentation  to  only  certain  monosaccharides  is,  accord- 
ing to  E.  Fischer,  like  the  action  of  the  inverting  enzymes  upon  disac- 
charides  and  glucosides,  dependent  upon  the  stereometric  configuration  of 
the  sugars  (see  Chapter  I).  This  difference  in  configuration  Is  not  only 
important  for  the  action  of  lower  living  organisms  upon  the  sugars,  but 
also  upon  the  behavior  of  the  sugars  within  higher  developed  organisms. 
Thus  the  investigations  of  Neuberg  and  Wohlgemuth  1  upon  arabinose 
and  Neuberg  and  Mayer  2  on  mannoses  have  shown  that  in  rabbits  the 
1-arabinose  and  the  d-mannose  are  much  better  utilized  than  d-  and  i-ara- 
binose  or  1-  and  i-mannose,  and  they  have  also  shown  that  the  lower  organ- 
isms have  the  tendency  of  decomposing  inactive  substances  into  their 
optically  active  components  to  a  much  higher  degree  than  the  higher 
organisms. 

By  the  action  of  lower  organisms  of  various  kinds  the  sugars  may  be 
made  to  undergo  fermentations  of  different  kinds,  such  as  lactic  acid  and 
butyric  acid  fermentation  and  mucilaginous  fermentation. 

The  simple  varieties  of  sugar  occur  in  part  in  nature  as  such,  already 
formed,  which  is  the  case  with  both  of  the  very  important  sugars,  dextrose 
and  lievulose.  They  also  occur  in  great  abundance  in  nature  as  more 
complex  carbohydrates  (di-  and  polysaccharides) ;  also  as  ester-like  com- 
binations with  different  substances,  as  so-called  glucosides. 

1  Zeitschr.  f.  physiol.  Chem.,  35. 

2  Ibid.,  37. 


PENTOSES.  89 

Among  the  groups  of  monosaccharides  known  at  the  present  time,  those 
containing  less  than  five,  and  more  thanjix  carbon  atoms  in  the  molecule 
have  no  great  importance  msbi^cTiemistrjr,  although  they  are  of  high  scien- 
tific interest.  Of  the  other  two  groups  the  hexoses  are  of  the  greatest 
importance,  because  in  the  past  only  those  carbohydrates  with  six  carbon 
atoms  were  considered  as  true  carbohydrates.  As  the  pentoses  have  been 
the  subject  of  considerable  biochemical  investigations  of  late,  they  will 
also  be  given  in  short. 

Pentoses  (C5H10O5). 

As  a  rule  the  pentoses  do  not  occur  as  such  in  nature,  but  are  formed  in 
the  hydrolytic  splitting  of  several  more  complex  carbohydrates,  the  so-called 
pentosan*  s,  especially  on  boiling  gums  with  dilute  mineral  acids.  The 
pentosanes  exist  very  widely  distributed  in  the  plant  kingdom  and  are 
especially  of  great  importance  in  the  building  up  of  certain  plant  con- 
stituents. The  pentoses  were  first  found  by  Salkowski  and  Jastrowitz 
in  the  animal  kingdom  in  the  urine  of  a  person  addicted  to  the  morphine 
habit,  and  later  by  Salkowski  and  others  in  normal  human  urine.  Small 
quantities  of  pentoses  have  been  detected  by  Kulz  and  Vogel  j  in  the 
urine  of  diabetics,  as  also  in  dogs  with  pancreas  diabetes  or  phlorhizin 
diabetes.  Pentose  has  also  been  found  by  Hammabsten  amongst  the 
cleavage  products  of  a  nucleoproteid  obtained  from  the  pancreas,  and 
seems  also,  according  to  the  observations  of  Blumenthal,  to  be  a  constit- 
uent of  nucleoproteids  of  various  organs  such  as  the  thymus,  thyroid, 
brain,  spleen,  and  liver.  In  regard  to  the  quantity  of  pentoses  found  in 
the  various  organs  we  must  refer  to  the  works  of  Grund  and  of  Bendix 
and   Ebsteix.2 

The  pentosanes  (Stone,  Slowtzoff)  as  well  as  the  pentoses  are  of  the 
greatest  importance  as  foods  for  herbivorous  animals.  In  regard  to  the 
value  of  the  pentoses,  the  researches  of  Salkowski,  Cremer,  Neuberg 
and  Wohlgemuth  3  upon  rabbits  and  hens  show  that  these  animals  can 
utilize  the  pentoses  and  indeed  can  use  them  as  glycogen  formers,  and  at 
the  same  time  they  showed  the  extent  to  which  the  pentoses  act  as  true 

1  Salkowski  and  Jastrowitz,  Centralbl.  f.  d.  med.  Wissensch.,  1892,  337  and  593; 
Salkowski,  Berl.  klin.  Wochenschr.,  1895;  Bial,  Zeitschr.  f.  klin.  Med.,  39;  Bial  and 
Blumenthal,  Deutsch.  med.  Wochenschr.,  1901,  No.  2;  Kulz  and  Vogel,  Zeitschr.  f. 
Biologie,  32. 

2  Ilammarsten,  Zeitschr.  f.  physiol.  Chem.,  19;  also  Salkowski,  Berl.  klin.  Wochen- 
schr., 1S95;  Blumenthal,  Zeitschr.  f.  klin.  Med.,  34;  Grund,  Zeitschr.  f.  physiol.  Chem., 
35;   Bendix  and  Ebstein,  Zeitschr.  f.  allgemein.  Physiol.,  2. 

3  Stone,  Amer.  Chem.  Journ.,  14;  Slowtzoff,  Zeitschr.  f.  physiol.  Chem.,  34;  Sal- 
kowski, 1.  c>,  Centralbl.;  Cremer,  Zeitschr.  f.  Biologie,  29  and  42;  Neuberg  and  Wohl- 
gemuth, 1.  c. 


90  THE  CARBOHYDRATES. 

or  only  indirect  producers  of  glycogen  (see  Chapter  VIII).  The  pentoses 
seem  to  be  absorbed  by  human  beings  and  in  part  utilized,  but  they  pass 
in  part  into  the  urine  even  when  small  quantities  are  taken.1 

The  natural  pentoses  are  reducing  aldoses  and  as  a  rule  do  not  belong 
to  the  sugars  fermentable  with  yeast.  Still,  the  observations  of  Salkowski, 
Bendix,  Schone  and  Tollens  seem  to  indicate  that  the  pentoses  are 
fermentable.2  They  are  readily  decomposed  by  putrefaction  bacteria. 
On  heating  with  hydrochloric  acid  they  yield  furfurol,  but  no  levulinic 
acid.  The  furfurol  passing  over  on  distilling  with  hydrochloric  acid  can 
be  detected  by  the  aid  of  aniline-acetate  paper,  which  is  colored  beauti- 
fully red  by  furfurol.  In  the  quantitative  estimation  we  can  use  the  method 
suggested  by  Tollens,  which  consists  of  converting  the  furfurol  in  the 
distillate  into  phloroglucide  by  means  of  phloroglucin  and  weighing  (see 
Tollens  and  Krober,  Grund,  Bendix  and  Ebstein).3  The  two  follow- 
ing pentose  reactions,  as  suggested  by  Tollens,  are  especially  applicable: 

The  orcin-hydrochloric  acid  test.  Mix  with  the  solution  or  the 
substance  introduced  into  water  an  equal  volume  of  concentrated  hydro- 
chloric acid,  add  some  orcin  in  substance,  and  heat.  In  the  presence 
of  pentoses  the  solution  becomes  reddish  blue,  then  bluish  green^and  on 
spectroscopic  examination  an  absorption-band  is  observed  between  C  and 
D.  If  it  is  cooled  partly  and  shaken  with  amyl  alcohol  a  bluish-green 
solution  which  shows  the  same  band  is  obtained. 

The  phloroglucin-hydrochloric  acid  test.  This  test  is  performed  in 
the  same  manner  as  the  above,  using  phloroglucin  instead  of  orcin.  The 
solution  becomes  cherry-red  on  heating  and  then  becomes  cloudy  and  hence 
a  shaking  out  with  amyl  alcohol  is  very  necessary.  The  red  amyl  alcohol 
solution  shows  an  absorption  band  between  D  and  E.  The  orcin  test  is 
better  for  several  reasons  than  the  phloroglucin  test  (Salkowski  and 
Neuberg4).  In  regard  to  the  use  of  these  tests  in  urine  examination  see 
Chapter  XV. 

Arabinoses.  The  pentose  isolated  by  Neuberg  5  from  human  urine 
's  i-arabinose.  It  can  be  Isolated  from  the  urine  as  diphenylhydrazone, 
from  which  the  arabinose  can  be  separated  by  splitting  with  formaldehyde. 
The  i-arabinose  is  crystalline,  has  a  sweetish  taste,  is  optically  inactive, 
and   melts   at    163-164°  C.     Its   diphenylhydrazone  melts    at   206°  C,    is 

1  See  Ebstein,  Virchow's  Arch.,  129;  Tollens,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29, 
1208;  Cremer,  1.  c. ;  Lindemann  and  May,  Deutsch.  Arch.  f.  klin.  Med.,  56;  Sal- 
kowski, Zeitschr.  f.  physiol.  Chem.,  30. 

2  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  30;  Bendix,  see  Chem.  Centralbl.,  1900,  1; 
Schone  and  Tollens,  ibid.,  1901,  1. 

*  Bendix  and  Ebstein,  1.  c,  which  contains  the  literature. 

*  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  27;   Neuberg,  ibid.,  31. 
6  Ber.  d.  d.  chem.  Gesellsch..  33. 


HBX0SE8.  91 

insoluble  in  cold  water  and  alcohol,  but  readily  soluble  in  pyridine.  The 
oeazone  melts  at  1G6-1680  C. 

The  dextrorotatory  1-arabinose  is  obtained  by  boiling  gum  arabic  or 
cherry  gum  with  dilute  sulphuric  acid.  The  d-arabinosc  is  prepared  synthet- 
ically. Both„  of  these  arabinoses  give  diphenylhydrazones  which  melt 
at  216-218° C. 

Xyloses.  The  only  pentose  thus  far  isolated  from  the  animal  tissues  is 
1-xylose,  obtained  by  Neuberg  l  from  the  pancreas  proteids,  and  is  identi- 
cal with  the  xylose  found  widely  distributed  in  the  plant  kingdom  and 
<  obtained  from  wood-gum  by  boiling  with  dilute  acids.  Xylose  is  crystalline, 
melts  at  153-154°  C,  dissolves  very  readily  in  water  but  with  difficulty  in 
alcohol,  is  faintly  dextrorotatory,  (a)D  =  +18.1°,  and  gives  a  phenylosazone 
which  melts  at  159-160°  C. 

Hexoses  (C8H1206). 

The  most  important  and  best-known  simple  sugars  belong  to  this  group, 
and  the  other  bodies  which  have  been  considered  as  carbohydrates  in  the 
past_(with  the  exception  of  arabinose  and  inosite)  are  anhydrides  of  this 
group.  Certain  hexoses,  such  as  dextrose  and  kevulose,  either  occur  in_ 
nature  already  formed  or  are  produced  by  the  hydrolytic  splitting  of  other 
more  complicated  carbohydrates  or  glucosides.  Others,  such  as  mannose 
or  galactose,  are  formed  by  the  hydrolytic  cleavage  of  other  natural 
products;  while  some,  on  the  contrary,  such  as  gulose,  talose,  and  others, 
are  obtained  only  by  artificial  means. 

All  hexoses,  as  also  their  anhydrides,  yield  laevulinic  acid,  C5H803, 
besides  formic  acid  and  humus  substances  on  boiling  with  dilute  mineral 
acids,  gome  of  the  hexoses  are  fermentable  with  yeast,,  while  the  artificially 
prepared  hexoses  are  not,  or  at  least  only  incompletely  and  with  great 
difficulty. 

Some  hexoses  are  aldoses,  while  others  are  ketoses.  Belonging  to  the 
first  groupiTwe  have  mannose,  dextrose,  gulose,  galactose,  and  talose, 
and  to.  the_other_  L-evulose,  and  possibly  also  sorbinose.  We  differen- 
tiate also  between  the  d,  1,  and  i  modifications;  for  instance,  d-,  1-,  and 
i-dextrose;  hence  the  number  of  isomers  js_yery  great. 

The  most  important  syntheses  of  the  carbohydrates  have  been  made  by 
E.  Fischer  and  his  pupils  chiefly  within  the  members  of  the  hexose  group. 
A  short  summary  of  the  syntheses  of  hexoses  is  given  below. 

The  first  artificial  preparation  of  a  sugar  was  made  by  Butlerow.  On 
treating  trioxymethylene,  a  polymer  of  formaldehyde,  with  lime-water  he  ob- 
tained a   faintly  sweetish   sirup   called   methylenitan,     Loew2  later  obtained  a 

1  Ber.  d.  d.  chem.  Gesellsch.,  35. 

'Butlerow,  Ann.  d.  Chem.  u.  Pharm.,  120;  Compt.  rend.,  53;  O.  Loew,  Journ.  f. 
prakt.  Chem.  (X.  F.),  33,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  20,  21,  22. 


92  THE  CARBOHYDRATES. 

mixture  of  several  sugars,  from  which  he  isolated  a  fermentable  sugar,  called 
methose,  by  condensation  of  formaldehyde  in  the  presence  of  bases.  The  most 
important  and  comprehensive  syntheses  of  sugar  have  been  preformed  by  E. 
Fischer.1 

The  starting-point  of  these  syntheses  is  a-acrose,  which  occurs  as  a  condensa- 
tion product  of  formaldehyde.  The  name  a-acrose  has  been  given  to  this  body 
because  it  is  obtained  from  acrolein  bromide  by  the  action  of  bases  (Fischer). 
It  is  also  obtained  admixed  with  (3-acrose  on  the  oxidation  of  glycerine  with 
bromine  in  the  presence  of  sodium  carbonate  and  treating  the  resulting  mixture 
with  alkali.  On  the  oxidation  with  bromine  a  mixture  of  glycerine  aldehyde, 
CH2OH.CH(OH).CHO,  and  dioxyacetone,  CH2(OH).CO.CH2OH,  is  obtained. 
These  two  bodies  may  be  considered  as  true  sugar-glyceroses  or  trioses.  It 
seems  as  if  a  condensation  to  hexoses  takes  place  on  treatment  with  alkalies. 

a-acrose  may  be  isolated  from  the  above  mixture  and  obtained  pure  by  first 
converting  it  into  its  osazone  and  then  retransforming  this  into  the  sugar,  a-acrose 
is  identical  with  i-laevulose.  With  yeast  one  half,  the  laevogyrate  d-lsevulose, 
ferments  while  the  dextrogyrate  1-lsevulose  remains.  The  i-  and  1-lsevulose 
may  be  prepared  in  this  way. 

On  the  reduction  of  a-acrose  we  obtain  a-acrite,  which  is  identical  with  i-man- 
nite.  On  oxidation  of  i-mannite  we  obtain  i-mannose,  from  which  only  1-mannose 
remains  on  fermentation.  On  further  oxidation  of  i-mannose  it  yields  i-mannonic 
acid.  The  two  active  mannonic  acids  may  be  separated  from  each  other  by 
the  fractional  crystallization  of  their  strychnine  or  morphine  salts.  The  two 
corresponding  mannoses  may  be  obtained  from  these  two  acids,  d-  and  1-mannonic 
acids,  by  reduction. 

d-lsevulose  is  obtained  from  d-mannose  by  the  method  given  on  page  85,  using 
the  osazone  as  an  intermediate  step.  The  d-  and  1-mannonic  acids  are  partly 
converted  into  d-  and  1-gluconic  acid  on  heating  with  quinoline,  and  d-  or  1-glucose 
is  obtained  on  the  reduction  of  these  acids;  1-glucose  is  best  prepared  from 
1-arabinose  by  means  of  the  cyanhydrin  reaction,  using  1-gluconic  acid  as  the 
intermediate  step.  The  combination  of  1-  and  d-gluconic  acid,  forming  i-glu- 
conic  acid,  yields  i-glucose  on  reduction. 

The  artificial  preparation  of  sugars  by  means  of  the  condensation  of  formalde- 
hyde has  received  special  interest  because,  according  to  Baeyer's  assimilation 
hypothesis  of  plants,  formaldehyde  is  first  formed  by  the  reduction  of  carbon 
dioxide,  and  the  sugars  are  produced  by  the  condensation  of  this  formaldehyde. 
Bokorny  2  has  shown,  by  special  experiments  on  algse  Spirogyra,  that  formalde- 
hyde sodium  sulphite  was  split  by  the  living  algse  cells.  The  formal dehyde  set  . 
free  is  immediately  condensed  to  carbohydrate  and  precipitated  as  starch. 

Among  the  hexoses  known  at  the  present  time  only  dextrose,  laevulose, 
and  galactose  are  really  of  physiological-chemical  interest;  therefore  the 
other  hexoses  will  only  be  incidentally  mentioned. 

Dextrose  (d-glucose) — glucose,  grape-sugar,  and  diabetic  sugar — 
occurs  abundantly  in  the  grape,  and  also,  often  accompanied  with  lrcvulose 
(d-f ructose) ,  in  honey,  sweet  fruits,  seeds,  roots,  etc.  It  occurs  in,  thlT 
human  and  animal  intestinal  tract  during  digestion,  also  in  small  quantities 
in  the  blood  and  lymph,  and  as  traces  in  other  animal  fluids  and  tissues. 
It  only  occurs  as  traces  in  urine  under  normal  conditions,  while  in  diabetes 
the  quantity  is  very  large.     It  is  formed  in  the  hydrolytic  cleavage  of 


1  Ber.  d.  deutsch.  chem.  Gesellsch.,  21,  and  1.  c,  page  83. 

2  Biolog.  Centralbl.,  12,  321  and  481. 


DEXTROSE.  93 

starch,  dextrin,  and  other  compound  carbohydrates,  as  also  in  the  splitting 
of  glucosides.  That  dextrose;  can  be  formed  from  proteids  in  the  animal 
body  follows  from  several  observations  and  especially  by  the  experience 
in  severe  forms  of  diabetes. 

Properties  of  Dextrose  Dextrose  crystallizes  sometimes  with  1  molecule 
of  water  of  crystallization  in  warty  masses  or  small  leaves  or  plates,  and 
sometimes  when  free  from  water  in  needles  or  prisms.  The  sugar  contain- 
ing water  of  crystallization  melts  even  below  100°  C.  and  loses  its  water 
of  crystallization  at  110°  C.  The  anhydrous  sugar  melts  at  146°  C,  and 
is  converted  into  glucosan,  C6H10O5,  at  170°  C.  with  the  elimination  of 
water.  On  strongly  heating  it  is  converted  into  caramel  and  then  de- 
composes. 

Dextrose  is  readily  soluble  in  water.  This  solution,  which  is  not  as 
sweet  as  a  cane-sugar  solution  of  the  same  strength^  is  dextrogyrate  and 
shows  strong  birotation.  The  specific  rotation  is  dependent  upon  the 
concentration  of  the  solution,  as  it  increases  with  an  increase  in  the  con- 
centration. A  10  per  cent  solution  of  anhydrous  glucose  can  be  taken  as 
52.74°  at  20°  C.1  Dextrose  dissolves  sparingly  in  cold,  but  more  freely 
in  boiling  alcohol.  100  parts  alcohol  of  sp.  gr.  0.837  dissolves  1.95  parts 
anhydrous  dextrose  at  17.5°  C.  and  27.7  parts  at  the  boiling  temperature 
(Anthon  2).     Dextrose  is  insoluble  in  ether. 

If  an  alcoholic  caustic-potash  solution  is  added  to  an  alcoholic  solution  of 
dextrose,  an  amorphous  precipitate  of  insoluble  sugar-potash  compound 
is  formed.  On  warming  this  compound  it  decomposes  easily  with  the 
formation  of  a  yellow  or  brownish  color,  which  is  the  basis  of  Moore's 
test.     Dextrose  forms  also  compounds  with  lime  and  baryta. 

Moore  's  Test.  If  a  dextrose  solution  is  treated  with  about  one  quarter 
of  its  volume  of  caustic  potash  or  soda  and  wanned,  the  solution  becomes 
first  yellow,  then  orange,  yellowish  brown,  and  lastly  dark  brown.  It  has 
at  the  same  time  a  faint  odor  of  caramel,  and  this  odor  is  more  pro- 
nounced on  acidification.3  — <^^ 

Dextrose  forms  several  crystallizable  combinations  with  NaCl,  of  which     -cd 
the  easiest  to  obtain  is  (C6Hli06)2.NaCl  +H20,  which  forms  large  colorless 
six-sided  double  pyramids  or  rhomboids  with  13.52  per  cent  NaCl. 

Dextrose  in  neutral  or  very  faintly  acid  (organic  acid)  solution  passes 
into  alcoholic  fermentation  with  beer-yeast,  C0H1JO6  =  2C2H5.OH +2C02. 
Besides  the  alcohol  and  carbon  dioxide  there  are  formed,  especially  at 
higher  temperatures,   small    quantities    of    homologous    alcohols    (amyl- 


1  For  further  information  see  Tollens,  Handbuch  der  Kohlenhydrate.,  2.  Aufl.,  44. 

2  Cited  from  Tollens'  Handbuch. 

s  In  regard  to  the  products  formed  in  this  reaction,  see  Framm,  Pfliiger's  Arch.,  64, 
and  especially  Gaud,  Compt.  rend.,  119. 


94  THE  CARBOHYDRATES. 

alcohol),  glycerine,  and  succinic  acid.  In  the  presence  of  acid  milk  or 
cheese, the  dextrose  passes,  especially  in  the  presence  of  a  base  such  as 
ZnO  or  CaC03,  into  lactic-acid  fermentation.  The  lactic  acid  may  then 
f'zrther  pass  into  butyric-acid  fermentation:  2C3H603  =  C4H802+2C02+4H. 

Dextrose  reduces  several  metallic  oxides,  such  as  copper  oxide,  bismuth 
oxide,  mercuric  oxide  in  alkaline  solutions,  and  the  most  important 
reactions  for  sugar  are  based  on  this  fact. 

Trommer's  test  is  based  on  the  property  that  dextrose  possesses  of  re- 
ducing cupric  hydrate  in  alkaline  solution  into  cuprous  oxide.  Treat  the 
dextrose  solution  with  about  £-£  vol.  caustic  soda  and  then  carefully  add 
a  dilute  copper-sulphate  solution.  The  cupric  hydrate  is  thereby  dis- 
solved, forming  a  beautiful  blue  solution,  and  the  addition  of  copper  sul- 
phate is  continued  until  a  very  small  amount  of  hydrate  remains  undis- 
solved in  the  liquid.  This  is  now  warmed  and  a  yellow  hydrated  suboxide 
or  red  suboxide  separates  even  below  the  boiling-point.  If  too  little  copper 
salt  has  been  added,  the  test  will  be  yellowish  brown  in  color,  as  in  Moore's 
test;  but  if  an  excess  of  copper  salt  has  been  added,  the  excess  of  hydrate  is 
converted  on  boiling  into  a  dark-brown  hydrate  which  interferes  with  the 
test.  To  prevent  these  difficulties  the  so-called  Fehling's  solution  may 
be  employed.  This  reagent  is  obtained  by  mixing  before  use  equal 
volumes  of  an  alkaline  solution  of  Rochelle  salt  and  a  copper-sulphate  solu- 
tion (see  Quantitative  Estimation  of  Sugar  in  the  Urine  in  regard  to  con- 
centration). This  solution  is  not  reduced  or  noticeably  changed  by  boiling. 
The  tartrate  holds  the  excess  of  cupric  hydrate  in  solution,  and  an  excess 
of  the  reagent  does  not  interfere  in  the  performance  of  the  test.  In  the 
presence  of  sugar  this  solution  is  reduced. 

Bottger-Almen's  test  is  based  on  the  property  dextrose  possesses  of 
reducing  bismuth  oxide  in  alkaline  solution.  The  reagent  best  adapted  for 
this  purpose  is  obtained,  according  to  Nylander's1  modification  of 
Almen's  original  teat,  by  dissolving  4  grms.  Rochelle  salt  in  100  parts  10  per 
cent  caustic-soda  solution  and  adding  2  gran,  bismuth  subnitrate  and 
digesting  on  the  water-bath  until  as  much  of  the  bismuth  salt  is  dissolved 
as  possible.  If  a  dextrose  solution  is  treated  with  about  y^  vol.,  or  with  a 
larger  quantity  of  the  solution  when  large  quantities  of  sugar  are  present, 
and  boiled  for  a  few  minutes,  the  solution  becomes  first  yellow,  then  yel- 
lowish brown,  and  finally  nearly  black,  and  after  a  time  a  black  deposit 
of  bismuth  (?)  settles. 

The  property  of  dextrose  of  reducing  an  alkaline  solution  of  mercury  on 
boiling  is  the  basis  of  Knapp's  reaction  with  alkaline  mercuric  cyanide  and 
of  Sachsse's  reaction  with  an  alkaline  potassium-mercuric  iodide 
:-olution. 

1  Zeitschr.  f.  physiol.  Chem.,  8. 


DEXTROSE.  95 

On  heating  with  piiexylhydrazixe  acetate  a  dextrose  solution  give-  a 
precipitate  consisting  of  fine  yellow  crystalline  needles  which  are  nearly 
insoluble  in  water  but  soluble  in  boiling  alcohol,  and  which  separate  again 
on  treating  the  alcoholic  solution  with  water.  The  crystalline  precipitate 
consists  of  phcnylglucosazojie  (sec  page  84).  This  compound  melts  when 
pure  at  204-205°  C,  dissolves  readily  in  pyridine  (0.25  grm.  in  1  grm.), 
and  precipitates  again  from  this  solution  as  crystals  on  the  addition  of 
benzene,  ligroin,  or  ether.  According  to  Neuberg  x  this  behavior  can  be 
used  in  the  purification  of  the  osazone.  With  bromphenylhydrazine  a 
phenylhydrazone  can  be  obtained  which  is  not  readily  soluble  in  water 
or  alcohol,  and  from  this  the  dextrose  can  be  split  off  by  formaldehyde. 

Dextrose  is  not  precipitated  by  a  lead-acetate  solution,  but  is  almost 
completely  precipitated  by  a  solution  of  ammoniacal  basic  lead-acetate. 
On  wanning  the  precipitate  becomes  flesh-color  or  rose-red  (Rubxer's 
reaction  2). 

If  a  watery  solution  of  dextrose  is  treated  with  benzoylchloride  and 
an  excess  of  caustic  soda,  and  shaken  until  the  odor  of  benzoylchloride 
has  disappeared,  a  precipitate  of  benzoic-acid  ester  of  dextrose  will  be  pro- 
duced which  is  insoluble  in  water  or  alkali  (Baumaxx  3) . 

If  2~1  c.  c.  of  a  dilute  watery  solution  of  dextrose  is  treated  with  a  few 
diops  of  a  10  per  cent  alcoholic  solution  (free  from  acetone)  of  a-naphthol, 
the  liquid  is  colored  a  beautiful  violet  on  the  addition  of  1-2  c.  c.  of  concen- 
trated sulphuric  acid  (Molisch  4).  This  reaction  depends  on  the  formation 
of  furfurol  from  the  sugar  by  the  action  of  the  sulphuric  acid. 

Diazobenzenk-sulpiionic  ACin  gives  with  a  dextrose  solution  made  alkaline 
with  a  fixed  alkali  a  red  color,  after  10-15  minutes  gradually  changing  to  violet. 
Orthonitrophenyl-propiolic  acid  yields  indigo  when  boiled  with  a  small 
quantity  of  dextrose  and  sodium  carbonate,  and  this  is  converted  into  indigo-white 
by  an  excess  of  sugar.  An  alkaline  solution  of  dextrose  is  colored  deep  red  on 
being  warmed  with  a  dilute  solution  of  picric  acid. 

A  more  complete  description  as  to  the  performance  of  these  several  tests 
will  be  given  in  detail  in  a  subsequent  chapter  (on  the  urine). 

Dextrose  is  prepared  pure  by  inverting  cane-sugar  by  the  following 
simple  method  of  Soxhlet  and  Tollexs,  being  a  modification  of 
ScHwrARz's5  method: 

Treat  12  litres  90  per  cent  alcohol  with  480  c.  c.  fuming  hydrochloric 
acid  and  warm  to  45-50°  C. ;  gradually  add  4  kilos  of  powdered  cane-sugar, 
and  allow  to  cool  after  2  hours,  when  all  the  sugar  will  have  dissolved  and 

•  Ber.  d.  d.  chem.  Gesellsch.,  32,  3384. 
1  Zeitschr.  f.  Biologie,  20. 

s  Ber.  d.  deutsch.  chem.  Gesellsch.,  19;  also  Kueny,  Zeitschr.  f.  physiol.  Chem.,  14, 
and  Skraup,  Wien.  Sitz.  Ber.,  98  (1888). 

*  Monatshefte  f.  Chem.,  7,  and  Centralbl.  f.  d.  med.  Wissensch.,  1887,  34  and  49. 
6  Tollens'  Handbuch  der  Kohlenhydrate,  2.  Aufl.,  39. 


96  THE  CARBOHYDRATES. 

been  inverted.  To  incite  crystallization,  some  crystals  of  anhydrous  dex- 
trose are  added,  and  after  several  days  the  crystals  are  sucked  dry  by 
the  air-pump,  washed  with  dilute  alcohol  to  remove  hydrochloric  acid,  and 
crystallized  from  alcohol  or  methyl  alcohol.  According  to  Tollens  it  is 
best  to  dissolve  the  sugar  in  one  half  its  weight  of  water  on  the  water- 
bath  and  then  add  double  this  volume  of  90-95  per  cent  alcohol. 

In  detecting  dextrose  in  animal  fluids  or  extracts  of  tissues  we  may 
make  use  of  the  above-mentioned  reduction  tests,  the  optical  determination, 
the  fermentation,  and  phenylhydrazine  tests.  For  the  quantitative  estima- 
tion the  reader  is  referred  to  the  chapter  on  urine.  Those  liquids  contain- 
ing proteids  must  first  have  these  removed  by  coagulation  with  heat  and 
addition  of  acetic  acid,  or  by  precipitation  with  alcohol  or  metallic  salts 
before  testing  for  dextrose.  In  regard  to  the  difficulties  of  operating  with 
blood  and  serous  fluids  we  refer  the  student  to  the  works  of  Schenck, 
Rohm ann,  Abeles,  and  Seegen.1 

The  guloses  are  stereoisomers  of  dextrose  and  may  be  prepared  artificially, 
■d-gulose  is  obtained  on  the  reduction  of  d-gulonic  acid,  which  is  derived  on  the 
reduction  of  glucuronic  acid. 

Mannoses. — d-mannose,  also  called  seminose,  is  obtained  with  d-laevulose 
on  the  careful  oxidation  of  d-mannite.  It  is  also  obtained  on  the  hydrolysis 
of  natural  carbohydrates,  such  as  salep  slime  and  reserve  cellulose  (especially 
from  the  shavings  from  the  ivory-nut).  It  is  dextrorotatory,  readily  ferments 
with  beer-yeast,  gives  a  hydrazone  not  readily  soluble  in  water,  and  an  osazone 
which  is  identical  with  that  from  d-glucose. 

Laevulose,  also  called  d-fructose,  fruit-sugar,  occurs,  as  above  stated, 
mixed  with  dextrose  extensively  distributed  in  the  vegetable  kingdom  and  also 
in  honey.  It  is  formed  in  the  hydrolytic  cleavage  of  cane-sugar  and  several 
other  carbohydrates,  but  it  is  readily  obtained  by  the  hydrolytic  splitting 
of  inulin.  In  extraordinary  cases  of  diabetes  mellitus  we  find  laevulose  in 
the  urine.  Neuberg  and  Strauss  2  have  detected  lsevulose  in  human  blood- 
serum  and  exudates  in  certain  cases  with  positiveness. 

Lsevulose  crystallizes  with  difficulty  in  needles  partly  anhydrous  and 
partly  containing  water.  It  is  readily  soluble  in  water,  but  nearly  insoluble 
in  cold  absolute  alcohol,  though  rather  readily  in  boiling  alcohol.  Its 
watery  solution  is  lsevogyrate.  Lsevulose  ferments  with  yeast,  and  gives 
the  same  reduction  tests  as  dextrose,  and  also  the  same  osazone.  It  gives  a 
combination  with  lime  which  is  less  soluble  than  the  corresponding  dextrose 
combination.  Lsevulose  is  not  precipitated  by  sugar  of  lead  or  basic  lead 
acetate. 

Lsevulose  does  not  reduce  copper  to  the  same  extent  as  dextrose. 
Under  similar  conditions  the  reduction  relationship  of  dextrose  to  lsevulose 
is  100  :  92.08. 

In  detecting  lsevulose  and  those  varieties  of  sugar  which  yield  lsevulose 


1  Schenck,  Pfluger's  Arch.,  4G  and  47;  Rohmann,  Centralbl.  f.  Physiol.,  4;  Abeles, 
Zeitschr.  f.  physiol.  Chem.,  15;  Seegen,  Centralbl.  f.  Physiol.,  4. 

2  Zeitschr.  f.  physiol.  Chem.,  36,  which  also  contains  the  older  literature. 


LWULOSE.  97 

on  cleavage  we  mako  use  of  the  following  reaction  suggested  by  Sf.li- 
WANOFP.  To  a  few  cubic  centimeters  of  fuming  hydrochloric  acid  and  -r — -/ 
an  equal  volume  of  water  add  a  small  quantity  of  the  sugar  solution  or 
of  the  solid  substance  and  a  few  crystals  of  resorcin  and  apply  heat.  The 
liquid  becomes  a  beautiful  red  and  gradually  a  substance  precipitates 
which  is  red  in  color  and  soluble  in  alcohol.  This  reaction,  which  accord- 
ing to  Rosin  may  be  made  more  delicate,  is,  as  Neuberg  *  has  shown, 
a  general  reaction  for  ketoses. 

According  to  Neuberg  2  methylphenylhydrazine  is  an  excellent  sub- 
stance to  use  for  the  separation  and  detection  of  laevulose,  as  it  gives 
a  characteristic  laevulose-methylphenylosazone.  This  osazone  when  re- 
crystallized  from  alcohol  melts  at  153°.  It  shows  a  dextrorotation  of 
1°  40'  when  0.2  grm.  of  the  osazone  are  dissolved  in  4  c.  c.  pyridine  and 
6  c.  c.  absolute  alcohol. 

Methylphenylhydrazine  is  a  very  excellent  means  for  separating  the 
aldoses  and  amino  sugar  from  the  ketoses.  The  aldoses  and  the  amino 
sugar  in  neutral  liquids  give  hydrazones  therewith,  and  after  the  removal 
of  these  the  osazones  of  the  ketoses  can  be  obtained  from  the  filtrate 
by  acidifying  with  acetic  acid  and  warming. 

Laevulose,  as  above  stated,  is  best  obtained  by  the  hydrolytic  cleavage 
of  inulin,  by  warming  with  faintly  acidulated  water. 

Sorbinose  (sorbin)  is  a  ketose  obtained  from  the  juice  of  the  berry  of  the 
mountain  ash  under  certain  conditions.  It  is  crystalline  and  is  laevogyrate, 
and  is  converted  into  sorbite  by  reduction. 

Galactose  (not  to  be  mistaken  for  lactose  or  milk-sugar)  is  obtained 
on  the  hydrolytic  cleavage  of  milk-sugar  and  by  hydrolysis  of  many  other 
carbohydrates,  especially  varieties  of  gums  and  slime  bodies.  It  is  also 
obtained  on  heating  cerebrin,  a  nitrogenized  glucoside  prepared  from  the 
brain,  with  dilute  mineral  acids. 

It  crystallizes  in  needles  or  leaves  which  melt  at  16S°  C.  It  is  some- 
what less  soluble  than  dextrose  in  water.  It  is  dextrogyrate  and  shows 
multirotation.  "With  ordinary  yeast  the  galactose  is  slowly,  but  neverthe- 
less completely,  fermented.  It  is  fermented  by  a  great  variety  of  yeasts 
(E.  Fischer  and  Thierfelder),  but  not  by  Saccharomyces  apiculatus,3 
which  is  of  importance  in  physiological-chemical  investigations.  Galactose 
reduces  Fehling  's  solution  to  a  less  extent  than  dextrose,  and  10  c.  c. 
of  this  solution  are  reduced,  according  to  Soxhlet,  by  0.0511  grm.  galactose 
in  1  per  cent  solution.  Its  phenylosazone  melts  at  193°  C,  and  is  soluble 
with  difficulty  in  water,  but  relatively  easy  in  hot  alcohol.  Its  solution 
in  glacial  acetic  acid  is  optically  inactive.     With  the  test  with  hydrochloric 

1  Zeitschr.  f.  physiol.  Chem.,  31;   Rosin,  ibid.,  3S. 

7  Ber.  d.  d.  chem.  Gesellsch.,  35;  also  Neuberg  and  Strauss,  ibid.,  36. 

8  See  F.  Voit,  Zeitschr.  f.  Biologie,  28  and  29. 


98  THE  CARBOHYDRATES. 

acid  and  phloroglucin  galactose  gives  a  color  similar  to  the  pentoses,  but 
the  solution  does  not  give  the  absorption  spectrum.  On  oxidation  it 
first  yields  galactonic  acid  and  then  mucic  acid.  Both  1-  and  i-galactose 
have  been  artificially  prepared. 

Talose  is  a  sugar  which  is  artificially  prepared  by  the  reduction  of  talonic 
acid.  Talonic  acid  is  obtained  from  d-galactonic  acid  by  heating  it  with  quinohne 
or  pyridine  to  140-150°  C. 

Appendix  to  the  Hexoses. 

CH2OH 

a-Glucosamine  *  (chitosamine),  C6H13N05  =  x',TTT,  whose  synthetical 

Ori.JNii2 

COH 
preparation  has  already  been  given  on  page  85,  was  first  prepared  by 
Ledderhose2  from  chitin  by  the  action  of  concentrated  hydrochloric  acid. 
Recently  it  has  been  obtained  as  a  cleavage  product  of  several  mucin 
substances  and  proteids  (see  pages  23  and  50).  Glucosamine  is,  as 
E.  Fischer  and  Leuchs  3  have  shown,  a  derivative  of  glucose  or  d-mannose 
(probably  dextrose),  and  as  an  intermediary  member  between  the  hexoses 
and  the  oxyamino  acids  obtainable  from  the  proteids,  it  forms  in  certain 
regards  a  bridge  between  the  proteids  and  the  carbohydrates. 

The  free  base  is  readily  soluble  in  water  with  an  alkaline  reaction  and 
quickly  decomposes.  The  characteristic  hydrochloride  forms  colorless 
crystals  which  are  stable  in  the  air  and  readily  soluble  in  water,  difficultly 
soluble  in  alcohol,  and  insoluble  in  ether.  The  solution  is  (o)d=  +70.15° 
to  74.64°  at  various  concentrations.4  Glucosamine  has  a  reducing  action 
similar  to  glucose,  gives  the  same  osazone,  but  is  not  fermentable.  With 
benzoyl  chloride  and  caustic  soda  it  gives  a  crystalline  ester.  In  alkaline 
solution  it  gives  with  phenylisocyanate  a  compound  which  can  be  con- 
verted into  its  anhydride  by  acetic  acid,  and  is  used  in  the  separation  and 
detection  of  glucosamine  (Steudel5).  On  oxidation  with  nitric  acid 
it  yields  norisosaccharic  acid,  whose  lead  salt  can  be  separated  and  whose 
salts  with  cinchonine  or  quinine  are  difficultly  soluble  in  water  and  can 
also  be  used  very  successfully  in  the  detection  of  glucosamine  (Neu- 
berg  and  Wolff  e).     On  oxidation  with  bromine  chitaminic  acid  (d-glucos- 

1  According  to  E.  Fischer's  suggestion  we  shall  use  the  term  glucosamine  instead 
of  the  term  chitosamine  which  has  lately  been  generally  used. 

2  Zeitschr.  f.  physiol.  Chem.,  2  and  4. 

3  Ber.  d.  d.  chem.  Gesellsch.,  36. 

4  See  Hoppe-Seyler-Thierf elder's  Handbuch,  7.  Aufl.;  Sundwik,  Zeitschr.  f.  physioL 
Chem.,  34. 

6  Zeitschr.  f.  physiol.  Chem.,  34. 
e  Ber.  d.  d.  chem.  Gesellsch. ,  34. 


GLUCURONIC  ACID.  99 

aminie  acid)  is  produced,  and  this  is  converted  into  chitaric  acid,  C8H10O8, 
by  nil miis  acid.  On  treatment  with  nitrous  acid  glucosamine  yields  a 
non-fermentable  sugar  called  chitose. 

Ehrlich  l  has  suggested  a  test  which  does  not  react  with  the  free  glucosamine, 
hut  with  the  mucins  and  other  protein  bodies  containing  an  acetylated  glucos- 
amine. It  consists  in  wanning  the  BUbstance,  which  lias  previously  been  treated 
with  alkali,  with    a    hydrochloric-acid   solution   of  dimethylaminobenzaldehyde, 

\vh  n  a  h  autiful  red  color  is  obtained. 

Glucosamine  h  host  prepared  from  decalcified  lobster-shells  by  treating 
with  hot  concentrated  hydrochloric  acid.2  In  regard  to  its  preparation 
from  protein  substances  we  must  refer  to  the  works  cited  on  page  23, 
foot-note  7. 

Galactosamine  has  been  prepared  by  Schulz  and  Ditthorn  3  from  a 
glucoproteid  of  the  proteid  glands  of  the  frog. 

CHO 
Glucuronic  acid  (glycuronic  acid).  C6Hi0O7=(CH.OH)<,  is  a  derivative 

COOH 

of  dextrose  and  has  been  synthetically  prepared  by  E.  Fischer  and 
Piloty  '  by  the  reduction  of  the  lactone  of  saccharic  acid.  On  oxidation 
with  bromine  it  forms  saccharic  acid,  and  on  reduction  it  yields  gulonic 
acid  lactone.  Salkowski  and  Neuberg  5  have  obtained  1-xylose  from 
glucuronic  acid  by  splitting  off  C02  by  means  of  putrefaction  bacteria. 

Glucuronic  acid  has  not  been  found  in  the  free  state  in  the  animal  body. 
It  occurs  to  a  slight  extent  in  normal  urine  as  a  conjugated  acid,  phenol,  and 
probably  also  indoxyl  and  skatoxyl  glucuronic  acid.  It  occurs  to  a  much 
greater  extent  in  urine  as  conjugated  acid  after  the  introduction  of  several 
aromatic  and  also  aliphatic  substances,  especially  after  camphor  and  chloral 
hydrate.  It  was  obtained  first  by  Schmiedeberg  and  Meyer  from  campho- 
glucuronic  acid  and  then  by  v.  Mering  6  from  urochloralic  acid  by  cleavage 
with  dilute  acids.  According  to  P.  Mayer  7  on  the  oxidation  of  dextr 
partial  formation  of  glucuronic  acid  and  oxalic  acid  takes  place,  and  there- 
fore, according  to  him,  an  increased  elimination  of  conjugated  glucuronic 
acids  show  in  certain  cases  an  incomplete  oxidation  of  dextrose.  Con- 
jugated glucuronic  acids  may  also  occur  in  the  blood  (P.  Mayer  8),  also 

1  Mediz.,  Woche  1901,  No.  15;  see  Langstein,  Ergebnisse  der  Physiol.,  I,  Abt.  I,  8S. 
1  See  Hoppe-Seyler-Thierfelder's  Handbuch,  7.  Aufl. 
s  Zeitschr.  f.  physiol.  Chera.,  29. 

4  Ber.  d.  d.  chem.  Gesellsch.    24. 

5  Zeitschr.  f.  physiol.  Chem.,  36. 

8  Mayer  and  Xeuberg,  Zeitschr.  f.  physiol.  Chem.,  29;  Schmiedeberg  u.  Meyer,  ibid., 
3;    v.  Mering,  ibid.,  6. 

7  Zeitschr.  f.  klin.  Med.,  47. 

8  Zeitschr.  f.  physiol.  Chem.,  32. 


100  THE  CARBOHYDRATES. 

in  the  faeces  and  bile.1  The  most  abundant  source  of  glucuronic  acid  is 
the  artist  pigment  "Jaune  indien,"  which  contains  the  magnesium  salt 
of  euxanthic  acid  (euxanthon-glucuronic  acid). 

Glucuronic  acid  is  not  crystalline,  but  is  only  obtainable  as  a  sirup. 
It  dissolves  in  alcohol  and  is  readily  soluble  in  water.  If  the  aqueous  solu- 
tion is  boiled  for  an  hour  the  acid  is  partly  (20  per  cent)  converted 
into  the  crystalline  lactone,  glucuron,  C6H806,  which  is  soluble  in  water 
and  insoluble  in  alcohol.  The  alkali  salts  of  the  acid  are  crystalline.  If 
a  concentrated  solution  of  the  acid  is  saturated  with  barium  hydrate  the 
basic  barium  salt  is  obtained  as  a  precipitate.  The  neutral  lead  salt  is 
soluble  in  water,  while  the  basic  salt  is  insoluble.  The  readily  crystallizable 
cinchonine  salt  can  be  used  in  isolating  glucuronic  acid  (Neuberg  2). 
Glucuronic  acid  is  dextrorotatory,  while  the  conjugated  acids  are  laevo- 
rotatory;  they  behave  like  dextrose  with  the  reduction  tests  and  do  not 
ferment  with  yeast.  They  give  the  pentose  reactions  with  phloroglucin  or 
orcin  and  hydrochloric  acid  and  yield  abundant  furfurol  on  distillation 
with  hydrochloric  acid.  With  the  phenylhydrazine  test  they  give  crystal- 
line compounds  which  are  not  sufficiently  characteristic  (Thierfelder, 
P.  Mayer3).  With  p-bromphenylhydrazine  hydrochloride  and  sodium 
acetate  they  give  p-bromphenylhydrazine  glucuronate,  which  is  charac- 
terized by  insolubility  in  absolute  alcohol  and  by  a  very  prominent  laevo- 
rotatory  action.  This  compound  is  very  well  suited  for  the  detection  of 
glucuronic  acid.4  Dissolved  in  a  mixture  of  alcohol  and  pyridine  (0.2  grm. 
substance  in  4  c.  c.  pyridine  and  6  c.  c.  alcohol)    the  rotation  is  =7°  25', 

20 
which  corresponds  to  (a)  —  =  —369°. 

Glucuronic  acid  is  best  prepared  from  euxanthic  acid,  which  decomposes 
by  heating  it  with  water  to  120°  C.  for  several  hours.  The  filtrate  from 
the  euxanthon  is  concentrated  at  40°  C,  when  the  anhydride  gradually 
crystallizes  out.  On  boiling  the  mother-liquor  for  some  time  and  re- 
evaporation  the  crystals  of  the  lactone  are  obtained. 

Disaccharides. 

Some  of  the  varieties  of  sugax-belonging  to  this  group  occur  ready 
formed  in  nature.  Thus  we  have  saccharose  and  lactose.  Some,  on  the 
contrary,  such  as  maltose  and  isomaltose,  are  produced  by  the  partial 
hydrolytic  cleavage  of  complicated  carbohydrates.  Isomaltose  is  besides 
this  also  obtained  from  dextrose  by  reversion  (see  next  page). 

1  See  Bial,  Hofmeister's  Beitriige,  3,  and  Leersura,  ibid. 

2  Ber.  d.  d.  chem.  Gesellsch.,  33. 

3  Thierfelder,  Zeitschr.  f.  physiol.  Chem.,  11,  13,  15;   P.  Mayer,  ibid.,  29. 

4  See  Neuberg,  Ber.  d.  d.  chem.  Gesellsch.,  32,  and  Mayer  and  Neuberg,  Zeitschr. 
f.  physiol.  Chem.,  29. 


DISACCIIARIDES.  101 

The  disaccharides  or  h< ■xobioses  _are  to  be  considered  as  anhydridesr 

derived  from   two  monosaccharides  with  tin-  exit   of   1   molecule  of  water. 
Corresponding  to  this,  their  general  formula  is  C12H22On.     On  hydrolytic 
cleavage  and  the  addition  of  water,  they  yield  two  molecules  of  hexoc 
and  indeed  either  two  molecules  of  the  same  hexose  or  two  different  hexoses. 
Thus  ,  M 

Saccharose + H,0  =  dextrose + lsevulose ; 

Maltose       -f  H20  =  dextrose  +  dextrose  ; 

Lactose       +H20  =  dextrose  4-  galactose. 

The  laevulose L_turgg_ the  polarized  raymore  to  the  left  than  the  dextrose 
does  to  the  right;  hence  the  mixture  of  hexoses  obtained  on  the  cleavage  of 
saccharose  has  an  opposite  rotation  to  the  saccharose  itself.  On  this 
account  the  mixture  is  called  invert-sugar,  and  the  hydrolytic  splitting^ 
is  designated  as  inversion.  This  term  inversion  is  not  only  used  for  the 
splitting  of  saccharose,  but  is  also  used  for  the  hydrolytic  cleavage  of 
compound  sugars  into  monosaccharides.  The  reverse  reaction,  whereby 
monosaccharides  are  condensed  into  complicated  carbohydrates,  is  called 
reversion. 

We  subdivide  the  disaccharides  into  two  groups:  first,  to  which  sac- 
charose belongs,  where  the  members  do  not  have  the  property  of  reducing 
certain  metallic  oxides;  and  the  second  group,  to  which  the  two  maltoses 
and  lactose  belong,  the  members  acting  like  monosaccharides  in  regard 
to  the  ordinary  reduction  tests.  The  members  of  this  last  group  have  the 
character  of  aldehyde-alcohols. 

Saccharose,  or  cane-sugar,  occurs  extensively  distributed  in  the  plant 
kingdom.  It  occurs  to  the  greatest  extent  in  the  stalk  of  the  sugar-millet 
and  sugar-cane,  the  roots  of  the  sugar-beet,  the  trunk  of  certain  varieties  of 
palms  and  maples,  in  carrots,  etc.  Cane-sugar  is  of  extraordinarily  great 
importance  as  a  food  and  condiment. 

Saccharose  forms  large,  colorless  monoclinic  crystals.  On  heating  it 
melts  in  the  neighborhood  of  ItiO3  C,  and  on  heating  more  strongly  it  turns 
brown,  forming  so-called  caramel.  It  dissolves  very  readily  in  water,  and 
according  to  Scheibleb  x  100  parts  saturated  saccharose  solution  contains  67 
parts  sugar  at  20°  C.  It  dissolves  with  difficulty  in  strong  alcohol.  Cane-_ 
sugar  is  strongly  dextrorotatoryT  The  specific  rotation  is  only  slightly 
modified  by  concentration,  but  is  markedly  changed  by  the  presence  of 
other  inactive  substances.     The  specific  rotation  is  (a)D=  +66.5°. 

Saccharose  acts  indifferently  towards  Moore  's  test  .and  to  the  ordinary 
reduction  tests.  It  does  not  ferment  directly,  but  only  after  inversion. 
which  can  be  brought  about  by  an  enzyme  (invertin)  contained  in  the  yeast. 
An  inversion  of  cane-sugar  also  takes  place  in  the  intestinal  canal.     Con- 


1  See  Tollens'  Handbuch  der  Kohlenhydrate,  2.  Aufl.,  124. 


102  THE  CARBOHYDRATES. 

centrated  sulphuric  acid  blackens  cane-sugar  very  quickly  even  at  the 
ordinary  temperature,  and  anhydrous  oxalic  acid  acts  the  same  on  warming 
on  the  water-bath.  Various  products  are  obtained  on  the  oxidation  of 
cane-sugar,  dependent  upon  the  variety  of  oxidizing  material  and  also  upon 
the  intensity  of  the  action.  Saccharic  acid  and  oxalic  acid  are  the  most 
important  products. 

The  reader  is  referred  to  complete  text-books  on  chemistry  for  the 
preparation  and  quantitative  estimation  of  cane-sugar. 

Maltose  (malt-sugar)  is  formed  in  the  hydrolytic  cleavage  of  starch  by 
malt  diastase,  saliva,  and  pancreatic  juice.  It  Is  obtained  from  glycogen 
under  the  same  conditions  (see  Chapter  VIII).  Maltose  is  also  produced 
transitorily  in  the  action  of  sulphuric  acid  on  starch.  Maltose  forms  the 
fermentable  sugar  of  the  potato  or  grain  mash,  and  also  of  the  beerwort. 

Maltose  crystallizes  with  1  molecule  water  of  crystallization  in  fine 
white  needles.  It  is  readily  soluble  in  water,  rather  easily  in  alcohol,  but 
insoluble  in  ether.  Its  solutions  are  dextrorotatory;  and  the  specific 
rotation  is  variable,  depending  upon  the  concentration  and  temperature, 
but  is  considerably  stronger  than  dextrose.1  Maltose  ferments  readily  and 
completely  with  yeast,  and  acts  like  dextrose  in  regard  to  the  reduction 
tests.  It  yields  phenylmaltosazone  on  warming  with  phenylhydrazine  for 
\\  hours.  This  phenylmaltosazone  melts  at  206°  C.  and  is  more  soluble 
than  the  glucosazone.  Maltose  differs  from  dextrose  chiefly  in  the  folio w- 
ing:  It  does  not  dissolveas  readily  in  alcohoL  has  a  stronger  dextrorota- 
tory power,  has  a  feebler  reducing  action  on  Fehlixg's  solution.  10  c.  c. 
Fehling's  solution  is,  according  to  Soxhlet,2  reduced  by  77.8  milligrams 
anhydrous  maltose  in  approximately  1  per  cent  solution. 

Isomaltose.  This  variety  of  sugar  is  produced  by  reversion,  as  has 
been  shown  by  Fischer,3  besides  dextrin-like  products,  by  the  action  of 
fuming  hydrochloric  acid  on  dextrose.  A  reformation  of  isomaltose  and 
other  sugars  from  dextrose  can  also  be  brought  about  by  means  of  yeast 
maltase  (Hill  and  Emmerling4).  It  is  also  formed,  besides  ordinary 
maltose,  in  the  action  of  diastase  on  starch  paste,  and  occurs  in  beer  and 
in  commercial  starch-sugar.  The  formation  of  isomaltose  in  the  hydrolysis 
of  starch  by  malt  diastase  has  been  denied  by  many  investigators  because 
they  considered  isomaltose  as  contaminated  maltose.5     It  is  also  produced, 


*See  Hoppe-Seyler-Thierfelders'  Handbuch,  7.  Aufl. 

2  Cited  from  Tollens'  Handbuch  der  Kohlenhydrate,  2.  Aufl.,  154. 

*.Ber.  d.  deutsch.  chem.  Gesellsch.,  23  and  28. 

*  Emmerling,  Ber.  d.  d.  chem.  Gesellsch.,  34;  Hill,  ibid.,  34,  and  1.  c,  foot-note 
2,  page  14. 

5  Prown  and  Morris,  Journ.  of  Chem.  Soc,  1895;  Chem.  News,  72.  See  also  Ost, 
T'lrich.  and  Jalowetz,  Kef.  in  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  Ling  and  Paker, 
Journ.  of  Chem.  Soc,  1895;  Pattivin,  Chem.  Centralbl.,  1899,  II,  1023. 


POLYSACCHARIDES.  103 

with  maltose,  by  the  action  of  saliva  or  pancreatic  juice  (Kulz  and  Vogel) 
or  blood-serum  (Rohmann  x)  on  starch. 

Isomaltose  dissolves  very  readily  in  water,  has  a  pronounced  sweetish 
taste,  and  docs  not  ferment,  or,  according  to  some,  only  very  slowly.  It  is 
dextrorotatory,  and  has  very  nearly  the  same  power  of  rotation  as  maltose. 
Isomaltose  is  characterized  by  its  osazone.  This  forms  fine  yellow  needles, 
which  begin  to  form  drops  at  140°  C.  and  melt  at  150-153°  C.  It  is  rather 
easily  soluble  in  hoi  water  and  dissolves  in  hot  absolute  alcohol  much  more 
readily  than  the  maltosazone.  Isomaltose  reduces  copper  as  well  as  bis- 
muth solutions. 

Lactose  (.milk-sugar).  As  this  sugar  occurs  exclusively  in  the  animal 
world,  in  the  milk  of  human  beings  and  animals,  it  will  be  treated  in  a 
following  chapter  (on  milk). 

Polysaccharides. 

If  we  exclude  the  hexotrioses  and  the  few  remaining  sugar-like  poly- 
saccharides, this  group  includes  a  great  number  of  very  complex  carbo- 
hydrates, which  occur  only  in  the  amorphous  condition  or  at  least  not  as 
crystals  in  the  ordinary  sense.  Unlike  the  bodies  belonging  to  the  other 
groups,  these  have  no  sweet  taste.  Some  are  soluble  in  water,  while 
others  swell  up  therein,  especially  in  warm  water,  and  finally  are  neither 
dissolved  nor  visibly  changed.  Polysaccharides  are  ultimately  converted 
into  monosaccharides  by  hydrolytic  cleavage. 

The  polysaccharides  (not  suirar-like)  are  ordinarily  divided  into  the 
following  chief  groups:  starch  group,  gum  and  vegetable-mucilage  group, 
and  cellulose  group. 

Starch  Group.  (C6H10O5)x. 

Starch,  amyltjm,  (C6H10O5)x.  This  substance  occurs  in  the  plant  king- 
dom very  extensively  distributed  in  the  different  parts  of  the  plant,  espe- 
ciall}'  as  reserve  food  in  the  seeds,  roots,  tubers,  and  trunk. 

Starch  is  a  white,  odorless,  and  tasteless  powder,  consisting  of  small 
granules,  which  have  a  stratified  structure  and  different  shape  and  size  in 
different  plants.  According  to  the  ordinary  opinion  the  starch-granules 
consist  of  two  different  substances,  starch  oraxulose  and  starch  cellu- 
lose, of  which  the  first  only  goes  into  solution  on  treatment  with  diastatic 
enzymes. 

Starch  is  considered  insoluble  in  cold  water.  The  grains  swell  up  in 
warm  water  and  burst,  yielding  a  paste.  Starch  is  insoluble  in  alcohol  and 
ether.  On  heating  starch  with  water  alone,  or  heating  with  glycerine  to 
190°  C,  or  on  treating  the  starch-sirains  with  6  parts  dilute  hydrochloric 

1  Kulz  and  Vogel,  Zeitschr.  f.  Biologie,  31 ;  Rohmann,  Centralbl.  f.  d.  med.  Wissensch., 
1893,  849. 


104  THE  CARBOHYDRATES. 

acid  of  sp.  gr.  1.07  at  ordinary  temperature  for  six  to  eight  weeks/  it  is- 
converted  into  soluble  starch  (amylodextrin,  am  dulin).  Soluble  starch  is 
also  formed  as  an  intermediate  step  in  the  conversion  of  starch  into  sugar  by 
dilute  acids  or  diastatic  enzymes.  Soluble  starch  may  be  precipitated  from 
very  dilute  solutions  by  baryta-water.2 

Starch-granules  swell  up  and  form  a  pasty  mass  in  caustic  potash  or 
soda.  This  mass  gives  neither  Moore's  nor  Trommer's  test.  Starch 
paste  does  not  ferment  with  yeast.  The  most  characteristic  test  for  starch 
is  the  blue  coloration  produced  by  iodine  in  the  presence  of  hydriodic  acid 
or  alkali  iodides.3  This  blue  coloration  disappears  on  the  addition  of 
alcohol  or  alkalies,  and  also  on  warming,  but  reappears  again  on  cooling. 

On  boiling  with  dilute  acids  starch  is  converted  into  dextrose.  In  the 
conversion  by  means  of  diastatic  enzymes  we  have  as  a  rule,  besides  dextrin, 
maltose,  and  isomaltose,  only  very  little  dextrose.  We  are  considerably  in 
the  dark  as  to  the  kind  and  number  of  intermediate  products  produced  in 
this  process  (see  Dextrins). 

Starch  may  be  detected  by  means  of  the  microscope  and  by  the  iodine 
reaction.  Starch  is  quantitatively  estimated,  according  to  Sachsse's 
method,4  by  converting  it  into  dextrose  by  hydrochloric  acid  and  then 
determining  the  dextrose  by  the  ordinary  methods. 

Inulin,  (C6H10O5)x +H20,  occurs  in  the  underground  parts  of  many 
compositae,  especially  in  the  roots  of  the  inula  helenium,  the  tubers  of  the 
dahlia,  the  varieties  of  helianthus,  etc.  It  is  ordinarily  obtained  from  the 
tubers  of  the  dahlia. 

Inulin  forms  a  white  powder  similar  to  starch,  consisting  of  sphseroid 
crystals,  which  are  readily  soluble  in  warm  water  without  forming  a  paste. 
It  separates  slowly  on  cooling,  but  more  rapidly  on  freezing.  Its  solutions 
are  Isevogyrate  and  are  precipitated  by  alcohol,  and  are  only  colored  yellow 
with  iodine.  Inulin  is  converted  into  the  Isevogyrate  monosaccharide 
lsevulose  on  boiling  with  dilute  sulphuric  acid.  Diastatic  enzymes  have 
no  or  very  slight  action  on  inulin.5 

Lichenin  (moss-starch)  occurs  in  many  lichens,  namely,  in  Iceland  moss. 
It  is  not  soluble  in  cold  water,  but  swells  up  into  a  jelly.  It  is  soluble  in  hot 
water,  forming  a  jelly  on  allowing  the  concentrated  solution  to  cool.  It  is  colored 
yellow  by  iodine  and  yields  glucose  on  boiling  with  dilute  acids.  Lichenin  is 
not  changed  by  diastatic  enzymes  such  as  ptyalin  or  amylopsin  (Nilson6). 

1  See  Tollens'  Handb.,  191.  In  regard  to  other  methods,  see  Wroblewski,  Ber.  d. 
deutsch.  chem.  Gesellsch.,  30;  Syniewski,  -ibid. 

2  In  regard  to  the  combinations  of  soluble  starch  and  dextrins  with  barium  hydrate, 
Bee  Biilow,  Pfluger's  Arch.,  (12. 

3  See  Mylius,  Ber.  d.  deutsch.  chem.  Gesellsch.,  20,  and  Zeitschr.  f.  physiol.  Chem.,  11. 
*  Tollens'  Handb.,  2.  Aufl.,  187. 

6  Ibid.,  208. 

6  Upsala  Lakaref.  Forh.,  28. 


GUMS  AND  MUCILAGES.  105 

Glycogen.  This  carbohydrate,  which  stands  to  a  certain  extent  between 
starch  and  dextrin,  is  principally  found  in  the  animal  kingdom,  hence  it 
will  be  considered  in  a  subsequent  chapter  (on  the  liver). 


The  Gums  and  Vegetable  Mucilages,  (C6H10O5)x. 

These  bodies  may  be  divided  into  two  chief  groups,  according  to  their 
origin  and  occurrence,  namely,  the  dextrin  group  and  the  vegetable  gums  or 
mucilages.  The  dextrins  stand  in  close  relationship  to  the  starches  and 
arc  formed  therefrom  as  intermediate  products  in  the  action  of  acids  and 
diastatic  enzymes.  The  various  kinds  of  vegetable  gums  and  vegetable  mu- 
cilages  occur,  on  the  contrary,  as  natural  products  in  the  vegetable  kingdom, 
and  some  may  be  separated  from  certain  plants  as  amorphous,  transparent 
masses  and  others  may  be  extracted  from  certain  parts  of  the  plant,  such 
as  the  wood  and  seeds,  by  proper  solvents. 

The  dextrins  yield  as  final  products  only  hexoses,  and  indeed  only 
dextrose  on  complete  hydrolysis.  The  vegetable  gums  and  the  mucilages 
yield,  on  the  contrary,  not  only  hexoses,  but  also  an  abundance  of  pentoses 
(gum  arabic  and  wood-gum),  d-galactose  occurs  often  amongst  the 
hexoses,  and  as  differentiation  from  the  dextrins  the}'  yield  mucic  acid  on 
oxidation  with  nitric  acid.  The  dextrins,  as  well  as  the  ordinary  varieties 
of  gums  and  mucilages,  are  precipitated  by  alcohol.  Basic  lead  acetate 
precipitates  the  gums  and  mucilages,  but  not  the  dextrins. 

Dextrin  (starch-gum,  British  gum)  is  produced  on  heating  starch  to 
200-210°  C,  or  by  heating  starch,  which  has  previously  been  moistened 
with  water  containing  a  little  nitric  acid,  to  100-110°  C.  Dextrins  are  also 
produced  by  the  action  of  dilute  acids  and  diastatic  enzymes  on  starch. 
Wc  arc  not  quite  clear  in  regard  to  the  steps  taking  place  in  the  above 
processes,  but  the  ordinary  views  are  as  follows:  Soluble  starch  is  the  first 
product  which  gives  a  blue  with  iodine,,  then  amylodcxtrin ,  which  on  further 
hydrolytic  cleavage  yields  sugar  and  erijthrodtxirin,  which  is  colored  red_ 
by  iodine.  On  further  cleavage  of  this  erythrodextrin  more  sugar  and 
a  dextrin,  achroodextrin,  which  is  not  colored  by  iodine,  is  formed.  From 
this  achroodextrin  after  successive  splittings  we  have  sugar  and  dextrins 
of  lower  molecular  weights  formed,  until  finally  we  have  sugar  and  a  dextrin, 
maltodcxtrin,  which  refuses  to  split  further,  as  final  products.  The  views 
are  rather  contradictory  in  regard  to  the  number  of  dextrins  which  occur 
as  intermediate  steps.  The  sugar  formed  is  isomaltose,  from  which  mal- 
tose and  only  very  little  dextrose  are  produced.  Another  view  is  that 
first  several  dextrins  are  formed  consecutively  in  the  successive  splitting 
with  hydration,  and  then  finally  the  sugar  is  formed  by  the  splitting  of 


106  THE  CARBOHYDRATES. 

the  last  dextrin.  Other  investigators,  especially  Syniewski,  have  recently 
suggested  views  on  this  subject.1 

The  various  dextrins  have  not  as  yet  been  separated  from  each  other, 
nor  isolated  as  chemical  individuals.  Recently  Young  2  has  tried  their 
separation  by  means  of  neutral  salts,  especially  ammonium  sulphate.  We 
cannot  enter  into  the  differences  as  to  the  dextrins  so  separated,  and  only 
the  characteristic  properties  and  reactions  will  be  given  for  the  dextrins  in 
general. 

The  dextrins  appear  as  an  amorphous,  white  or  yellowish-white  powder 
which  is  readily  soluble  in  water.  Their  concentrated  solutions  are  viscid 
and  sticky,  similar  to  gum  solutions.  The  dextrins  are  dextrogyrate. 
They  are  insoluble  or  nearly  so  in  alcohol,  and  insoluble  in  ether.  Watery 
solutions  of  dextrins  are  not  precipitated  by  basic  lead  acetate.  Dextrins 
dissolve  cupric  hydrate  in  alkaline  liquids,  forming  a  beautiful  blue  solu- 
tion, which,  as  is  generally  admitted,  is  reduced  by  pure  dextrins.  The 
dextrins  are  not  directly  fermentable.. 

The  vegetable  gums  are  soluble  in  water,  forming  solutions  which  are  viscid 
but  may  be  filtered.  We  designate,  on  the  contrary,  as  vegetable  mucilages 
those  varieties  of  gum  which  do  not  or  only  partly  dissolve  in  water,  and  which 
swell  up  therein  to  a  greater  or  less  extent.  The  natural  varieties  of  gum  and 
mucilage,  to  which  several  generally  known  and  important  substances,  such  as 
gum  arabic,  wood-gum,  cherry-gum,  salep,  and  quince  mucilage,  and  probably 
also  the  little-studied  pectin  substances  belong,  will  not  be  treated  in  detail, 
because  of  their  unimportance  from  a  physiological  standpoint. 

The  Cellulose  Group  (C6H10O5)x. 

Cellulose  is  that  carbohydrate,  or  perhaps  more  correctly  mixture  of 
carbohydrates,  which  forms  the  chief  constituent  of  the  walls  of  the  plant- 
cells.  This  is  true  for  at  least  the  walls  of  the  young  cells,  while  in  the 
walls  of  the  older  cells  the  cellulose  is  extensively  incrusted  with  a  sub- 
stance called  lignin. 

The  true  celluloses  are  characterized  by  their  great  insolubility.  They 
are  insoluble  in  cold  or  hot  water,  alcohol,  ether,  dilute  acids,  and  alkalies. 
We  have  only  one  specific  solvent  for  cellulose,  and  that  is  an  ammoniacal 
solution  of  copper  oxide  called  Schweitzer's  reagent.  The  cellulose  may 
be  precipitated  from  this  solvent  by  the  addition  of  acids,  and  obtained  as 
an  amorphous  powder  after  washing  with  water. 

1  In  regard  to  the  various  views  on  the  theories  of  the  saccharification  of  Starch, 
see  Musculus  and  Gruber,  Zeitschr.  f.  physiol.  Chem.,  2;  Lintner  and  Dull,  Ber.  d.  d. 
chem.  Gescllsch.,  20  and  28;  Biilow,  1.  c. ;  Brown  and  Heron,  Journ.  of  chem.  Soc,  1S79; 
Brown  and  Morris,  ibid.,  1885  and  1889;  Syniewski,  Annal.  d.  Chem.  u.  Pharm.,  309,  and 
Chem.Centralbl.,  1902,  2. 

2  Journ.  of  Physiol.,  22,  which  contains  the  older  researches  of  Nasse,  Kriiger, 
Neunicistcr,  Pohl,  and  Halliburton. 


CELLULOSES.  107 

Cellulose  is  converted  into  a  substance,  so-called  amyloid,  which  gives 
a  blue  coloration  with  iodine  by  the  action  of  concentrated  sulphuric  acid. 
By  the  action  of  strong  nitric  acid  or  a  mixture  of  nitric  acid  and  concen- 
trated sulphuric  acid  celluloses  are  converted  into  nitric-acid  esters  or  oitro- 
celluloses,  which  are  highly  explosive  and  have  found  great  practical  use. 

The  ordinary  celluloses  when  treated  at  the  ordinary  temperature  with 
strong  sulphuric  acid  and  then  boiled  for  some  time  after  diluting  with 
water  are  converted  into  dextrose.  We  also  have  celluloses  which  behave 
differently,  namely  those  which  yield  mannose  on  the  above  treatment. 

Hemicelluloses  are,  according  to  E.  Schulze,  those  constituents  of  the 
cell-wall  related  to  cellulose  which  differ  from  the  ordinary  cellulose  by  dissolv- 
ing on  heating  with  strongly  diluted  mineral  acids,  such  as  1.25  per  cent  sulphuric 
acid,  and  of  yielding  arabinose,  xylose,  galactose,  and  mannose  instead  of  dextrose. 
The  hemicelluloses  (from  lupin  seeds)  are  hydrolized  even  by  0.1  per  cent  hydro- 
chloric acid  and  are  dissolved,  although  only  slowly,  by  diastatic  enzymes  (Schulze 
and  Castoro  *)• 

The  cellulose,  at  least  in  part,  undergoes  decomposition  in  the  intestinal 
tract  of  man  and  animals.  A  closer  discussion  of  the  nutritive  value  of 
cellulose  will  be  given  in  a  future  chapter  (on  digestion).  The  great  impor- 
tance of  the  carbohydrates  in  the  animal  economy  and  to  animal  metab- 
olism will  also  be  given  in  the  following  chapters. 

1  E.  Schulze,  Zeitschr.  f.  physiol.  Chein.,  16  and  19,  with  Castoro,  ibid.,  36. 


CHAPTER  IV. 
THE  ANIMAL  FATS. 

The  fats  form  the  third  chief  group  of  the  organic  foods  of  man  and 
animals.  They  occur  very  widely  distributed  in  the  animal  and  plant 
kingdoms.  Fat  occurs  in  all  organs  and  tissues  of  the  animal  organism, 
though  the  quantity  may  be  so  variable  that  a  tabular  exhibit  of  the  amount 
of  fat  in  different  organs  is  of  little  interest.  The  marrow  contains  the 
largest  quantity,  having  over  96  per  cent.  The  three  most  important 
deposits  of  fat  in  the  animal  organism  are  the  intermuscular  connective 
tissue,  the  fatty  tissue  in  the  abdominal  cavity,  and  the  subcutaneous  con- 
nective tissues.  In  plants  the  seeds  and  fruit,  and  in  certain  instances 
also  the  roots,  are  rich  in  fat. 

The  fats  consist  nearly  entirely  of  so-called  neutral  fats  with  only  very- 
small  quantities  of  fatty  acids.  The  neutral  fats  are  esters  of  the  triatomic 
alcohol,  glycerine,  with  monobasic  fatty  acids.  These  esters  are  triglycerides, 
that  is,  the  hydrogen  atoms  of  the  three  hydroxyl  groups  of  the  glycerine 
are  replaced  by  the  fatty-acid  radicals,  and  their  general  formula  is  there- 
fore C3H5.O3.R3.  The  animal  fats  consist  chiefly  of  esters  of  the  three  fatty 
acids,  stearic,  palmitic,  and  oleic  acids.  In  certain  fats,  especially  in  milk- 
fat,  glycerides  of  fatty  acids  such  as  butyric,  caproic  caprylic,  and  capric 
acids  also  occur  in  considerable  amounts.  Besides  the  above-mentioned 
ordinary  fatty  acids,  stearic,  palmitic,  and  oleic  acids,  we  also  find  in  human 
and  animal  fat,  exclusive  of  certain  fatty  acids  only  little  studied,  the  fol- 
lowing non- volatile  fatty  acids,  as  glycerides,  namely,  lauric  acid,  C12H2402, 
myristic  acid,  CliK2S02,  and  arachidic  acid,  C20H40O2.  .  In  the  plant  kingdom 
triglycerides  of  other  fatty  acids,  such  as  lauric  acid,  myristic  acid,  linoleic 
acid,  erucic  acid,  etc.,  sometimes  occur  abundantly.  Besides  these,  oxy- 
acids  and  high  molecular  alcohols  have  been  found  in  many  plant  fats. 
The  occurrence  of  traces  of  these  oxyacids  in  the  animal  kingdom  has 
not  been  thoroughly  investigated,  but  the  occurrence  of  monoxystearic  acid 
seems  to  have  been  proven.1  The  occurrence  of  high  molecular  alcohols, 
although  ordinarily  only  in  small  amounts,  has  on  the  contrary  been  posi- 
tively shown  in  animal  fat. 

The  animal  fats  are  of  the  greatest  interest  and  consist  of  a  mixture  of 

1  Erben,  Zeitschr.  f.  physiol.  Chem.,  30;  Bernert,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 

108 


XKUTRAL  FATS.  109 

varying  quantities  of  tristeariNj  tbipalmitin,  and  triolein',  having  an 
average  elementary  composition  of  C  76.5,  H  12.0,  and  O  11.5  per  cent. 
It  must  be  remarked  that  in  animal  fat  (mutton  and  beef  tallow)  as  well 
as  in  plant  fat  (olive-oil)  mixed  triglycerides^  such  as  dipalmito-olein, 
distearo-palmitin.  distearo-olein,  occur  and  that  these  mixed  glycerides  may 
also  be  prepared  synthetically.1 

Fats  fmm  different  species  of  animals,  and  even  from  different  parts  of 
the  same  animal,  have  an  essentially  different  consistency,  depending  upon 
the  relative  amounts  of  the  different  fats.  In  solid  fats— as  tallow — 
tristearin  and  tripalmitin  are  in  excess,  while  the  less  solid  fats  are  charac- 
terized by  a  greater  abundance  of  tripalmitin  and  triolein.  This  last- 
mentioned  fat  is  found  in  greater  quantities  proportionally  in  cold-blooded 
animals,  and  this  accounts  for  the  fat  of  these  animals  remaining  fluid  at 
temperatures  at  which  the  fat  of  warm-blooded  animals  solidifies.  Human 
fat  from  different  organs  and  tissues  contains,  in  round  numbers,  G7-S5  per 
cent  triolein.2  The  melting-point  of  different  fats  depends  upon  the  com- 
position of  the  mixtures,  and  it  not  only  varies  for  fat  from  different  tissues 
of  the  same  animal,  but  also  for  the  fat  from  the  same  tissues  in  various 
kinds  of  animals. 

Neutral  fats  are  colorless  or  yellowish  and,  when  perfectly  pure,  odorless 
and  tasteless.  They  are  lighter  than  water,  on  which  they  float  when  in  a 
molten  condition.  They  are  insoluble  in  water,  dissolve  in  boiling  alcohol, 
but  separate  on  cooling, — often  in  crystals.  They  are  easily  soluble  in 
ether,  benzene,  and  chloroform.  The  fluid  neutral  fats  give  an  emulsion 
when  shaken  with  a  solution  of  gum  or  albumin.  With  water  alone  they 
give  an  emulsion  only  after  vigorous  and  prolonged  shaking,  but  the 
emulsion  i-^  not  persistent.  The  presence  of  some1  soap  causes  a  very  fine 
and  permanent  emulsion  to  form  easily.  Fat  produces  spots  on  paper 
which  do  not  disappear;  it  is  not  volatile;  it  boils  at  about  300°  C.  with 
partial  decomposition,  and  burns  with  a  luminous  and  smoky  flame.  The 
fatty  acids  have  most  of  the  above-mentioned  properties  in  common  with 
the  neutral  fats,  but  differ  from  them  in  being  soluble  in  alcohol-ether,  in 
having  an  acid  reaction,  and  by  not  giving  the  acrolein  test.  The  neutral 
fats  generate  a  strong  irritating  vapor  of  acrolein,  due  to  the  decomposition 
of  glycerine,  C3H3(OH)3  — 2HaO  =  C3H40;  when  heated  alone,  or  more 
easily  when  heated  with  potassium  bisulphate  or  with  other  dehydrating' 
substances. 

The  neutral  fats  may  be  split  by  the  addition  of  the  constituents  of 

1  Guth,  Zeitschr.  f.  Biologie,  44;  W.  Hansen,  Arch.  f.  Hygiene,  42;  Holde  and 
Stange,  Ber.  d.  d.  chem.  Gesellsch.,  34;   Kreis  and  Hafner,  ibid.,  36. 

2  See  Knopf elmacher,  "Untersuch.  iiber  das  Fett  im  Siiuglingsalter, "  etc.,  Jahrbuch 
f.  Kinderheilkunde  (X.  F.),  45,  which  also  contains  the  older  literature;  Jaeckle, 
Zeitschr.  f.  physiol.  Chem.,  36. 


110  THE  ANIMAL  FATS. 

water  according  to  the  following  equation:  C3H5(OR)3+3H20  =  C3H5(OH)a 
+3HOR.  This  splitting  may  be  produced  by  the  pancreatic  enzyme  and 
other  enzymes  occurring  in  the  animal  and  vegetable  kingdoms,  or  by 
superheated  steam.  "We  most  frequently  decompose  the  neutral  fats  by 
boiling  them  with  not  too  concentrated  caustic  alkali,  or,  still  better  (in 
biochemical  researches),  with  an  alcoholic  potash  solution  or  with  sodium 
alcoholate.  By  this  procedure,  which-is  called  saponification,  the  alkali  salts 
of  the  fatty  acids  (soaps)  are  formed.  '  If  the  saponification  is  made  with 
load  oxide,  then  lead  plaster,  the  lead  salt  of  the  fatty  acids  is  produced. 
By  saponification  is  to  be  understood  not  only  the  cleavage  of  neutral  fats 
by  alkalies,  but  also  the  splitting  of  neutral  fats  into  fatty  acids  and  glycer- 
ine in  general. 

On  keeping  fats  for  a  long  time  in  contact  with  air  they  undergo  a  change, 
becoming  yellow  in  color,  acid  in  reaction,  and  develop  an  unpleasant 
odor  and  taste.  They  become  rancid,  and  in  this  change  a  part  of  the 
fat  is  split  into  fatty  acids  and  glycerine,  and  then  an  oxidation  of  the  free 
fatty  acids  takes  place,  producing  volatile  bodies  of  an  unpleasant  odor. 

The  three  most  important  fats  of  the  animal  kingdom  are  stearin,. 
palmitin}  and  olein^ 

CH2.O.C18H350 

Stearin    or   tristearin,    C57H110O6  =  CH.O.C18H35O,    occurs    especially    in 

CH2.O.C18H350 
the  solid  varieties  of  tallows,  but  also  in  the  vegetable  fats.  Staaric  acid, 
C\8H3602,  is  found  in  the  free  state  in  decomposed  pus,  in  the  expectora- 
tions in  gangrene  of  the  lungs,  and  in  cheesy  tuberculous  masses.  It 
occurs  as  lime-soap  in  excrements  and  adipocere,  and  in  this  last  product 
also  as  an  ammonium  soap.  It  also  exists  as  alkali  soap  in  the  blood,  bile, 
transudations,  and  pus,  and  in  the  urine  to  a  slight  extent. 

Stearin  is  the  hardest  and  most  insoluble  of  the  three  ordinary  neutral 
fats.  It  is  nearly  insoluble  in  cold  alcohol,  and  soluble  with  great  difficulty 
in  cold  ether  (225  parts).  It  separates  from  warm  alcohol  on  cooling  as 
rectangular,  less  frequently  as  rhombic  plates.  The  statements  in  regard 
to  the  melting-point  are  somewhat  varied.  Pure  stearin,  according  to 
Heixtz,1  melts  transitorily  at  55°  and  permanently  at  71.5°.  The  stearin 
from  the  fatty  tissues  (not  pure)  melts  at  63°  C. 
CH3 

Stearic  acid,  (CH2)J6,  crystallizes  (on  cooling  from  boiling  alcohol)  in 
COOH 
large,  shining,  long  rhombic  scales  or  plates.     It  is  less  soluble  than  the 
other  fatty  acids  and  melts  at  69.2°  C.     Its  barium  salt  contains  19.49  per 
cent  barium,  and  its  silver  salt  contains  27.59  per  cent  silver. 


1  Annal.  d.  Chem.  u.  Pharm.,  92. 


PALMITIN  AND  0LE1N.  Ill 

C5Hf.0.CltHtt0 

Palmitin,  or  tripalmitin,  (,51lIu808  =  CH.O.('1(1II.tl(  >.     Of  the  two  solid  va- 

CH2.O.C10H31O 
rietics  of  fats,  palmitin  is  the  one  which  occurs  in  predominant  quantities 
in  human  fat  (Langer  ')  Palmitin  is  present  in  all  animal  fats  and  in 
several  kinds  of  vegetable  fat.  A  mixture  of  stearin  and  palmitin  was 
formerly  called  marc;arin.  As  to  the  occurrence  of  palmitic  acid,C1BII.r/)2, 
about  the  same  remarks  apply  as  to  stearic  acid.  The  mixture  of  these 
two  acids  has  been  called  margaric  acid,  and  this  mixture  occurs — often 
as  very  long,  thin,  crystalline  plates — in  old  pus,  in  expectorations  from 
gangrene  of  the  lungs,  etc. 

Palmitin.  crystallizes,  on  cooling  from  a  warm  saturated  solution  in  ether 
or  alcohol,  in  starry  rosettes  of  fine  needles.  The  mixture  of  palmitin  and 
stearin,  called  margarin,  crystallizes,  on  cooling  from  a  solution,  as  balls  or 
round  masses  which  consist  of  short  or  long,  thin  plates  or  needles  which 
often  appear  like  blades  of  grass.  Palmitin,  like  stearin,  has  a  variable 
melting  and  solidifying  point,  depending  upon  the  way  it  has  been  pre- 
viously treated.  The  melting-point  is  often  given  as  62°  C.  According 
to  other  statements,2  it  melts  at  50.5°  C,  solidifies  on  further  heating,  and 
melts  again  at  66.50°  C. 
CH, 

Palmitic  acid,  (CH2)H,  crystallizes  from  an  alcoholic  solution  in  tufts 
COOH 
of  fine  needles.  It  melts  at  62°  C.J  still  the  admixture  with  stearic  acid, 
as  Heixtz  has  shown,  essentially  changes  the  melting-  and  solidify ing-points 
according  to  the  relative  amounts  of  the  two  acids.  Palmitic  acid  is  some- 
what more  soluble  in  cold  alcohol  than  stearic  acid ;  but  they  have  about 
the  same  solubility  in  boiling  alcohol,  ether,  chloroform,  and  benzene.  Its 
barium  salt  contains  21.17  per  cent  barium,  and  the  silver  salt  contains 
29.72  per  cent  silver. 

CH2.O.C18H330 

Olein,  or  triolein,  C57H104O6  =  CH.O.C18H33O,  is  present    in    all   animal 

CHa.O.C18H„0 
fats,  and  in  greater  quantities  in  vegetable  fats.     It  is  a  solvent  for  stearin 
and  palmitin.     The  oleic  acid  (elaic  acid),  C18H3402,  has  as  soaps  probably 
the  same  occurrence  as  the  other  fatty  acids. 

<  )lein  is,  at  ordinary  temperatures,  a  nearly  colorless  oil  of  a  specific 
gravity  of  0.014,  without  odor  or  marked  taste,  and  solidifies  in  crystalline 
needles  at  —  6°  C.     It  becomes  rancid  quickly  if  exposed  to  the  air.     It 


1  Monatshefte  f.  Chem.,  2;  see  also  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  3G. 
1  R.  Benedikt,  Analyse  der  Fette.     3.  Aufl.,  1897,  44. 


112  THE  ANIMAL  FATS. 

dissolves  with  difficulty  in  cold  alcohol,  but  more  easily  in  warm  alcohol 
or  in  ether.     It  is  converted  into  its  isomer,  elaidin,  by  nitrous  acid. 

(CH2)7 
Oleic  acid,    ^^      ,  forms  on  heating,  besides  volatile  acids,  sebacic  acid, 

(CH2)7 
COOH 
C10H18O4,  which  crystallizes  in  shining  leaves  and  melts  at  127°  C.  With 
nitrous  acid  oleic  acid  is  transformed  into  the  isomeric  solid  elaidic  acid, 
which  melts  at  45°  C.  Oleic  acid  forms  at  ordinary  temperature  a  colorless, 
tasteless,  and  odorless  oily  liquid  which  solidifies  in  crystals  at  about 
4°  C,  which  then  melt  again  at  14°  C.  Oleic  acid  is  insoluble  in  water, 
but  dissolves  in  alcohol,  ether,  and  chloroform.  With  concentrated  sul- 
phuric acid  and  some  cane-sugar  it  gives  a  beautiful  red  or  reddish-violet 
liquid  whose  color  is  similar  to  that  produced  in  Pettenkofer's  test  for 
bile-acids.  Oleic  acid  is  an  unsaturated  fatty  acid  which  can  take  up 
halogens.  On  heating  with  hydriodic  acid  and  amorphous  phosphorus 
it  takes  up  hydrogen  and  is  converted  into  stearic  acid.  Oleic  acid  readily 
oxidizes  in  the  air,  yielding  acid  products.  The  monoxystearic  acid  found 
in  certain  animal  fats  may  be  formed  from  oleic  acid  by  oxidation.  The 
barium  salt  of  oleic  acid  contains  19.65  per  cent  barium  and  the  silver 
salt  27.73  per  cent  silver. 

If  the  watery  solution  of  the  alkali  combinations  of  oleic  acid  is  precipi- 
tated with  lead  acetate,  a  white,  tough,  sticky  mass  of  lead  oleate  is 
obtained  which  is  not  soluble  in  water  and  only  slightly  in  alcohol,  but  is 
soluble  in  ether.  This  salt  is  more  easily  soluble  in  benzene  than  the  lead 
salts  of  stearic  and  palmitic  acids,  and  this  behavior  of  the  lead  salts  towards 
ether  and  benzene  is  made  use  of  in  separating  oleic  acid  from  the  other 
fatty  acids. 

An  acid  related  to  oleic  acid,  doeglic  acid,  which  is  solid  at  0°  C,  liquid  at 
16°  C,  and  soluble  in  alcohol,  is  found  in  the  blubber  of  the  Baloena  rostrata. 
Kurbatoff  *  has  demonstrated  the  presence  of  linoleic  acid  in  the  fat  of  the  silurus, 
Sturgeon,  seal,  and  certain  other  animals.  Drying  fats  have  also  been  found  by 
Amthor  and  Zink  2  in  hares,  wild  rabbits,  wild  boar,  and  mountain-cock. 

To  detect  the  presence  of  fat  in  an  animal  fluid  or  tissue  the  fat  must 
first  be  shaken  out  or  extracted  with  ether.  After  the  evaporation  of  the 
ether  the  residue  is  tested  for  fat  and  the  acrolein  test  must  not  be  neg- 
lected. If  this  test  gives  positive  results,  then  neutral  fats  are  present; 
if  the  results  are  negative,  then  only  fatty  acids  are  present.  If  the  above 
residue  after  evaporation  gives  the  acrolein  test,  then  a  small  portion  is 
dissolved  in  alcohol-ether  free  from  acid  and  which  has  been  colored  bluish 

1  Maly's  Jahresber.,  22.  2  Zeitschr.  f.  analyt.  Chem.,  36. 


EXAMINATION  OF  FATS.  113 

violet  by  tincture  of  alkanet.  If  the  color  becomes  red,  a  mixture  of  neu- 
tral fat  and  fatty  acids  is  present.  In  this  case  the  fat  is  treated  while 
warm  with  a  soda  solution  and  evaporated  on  the  water-bath,  constantly 
stirring  until  all  the  water  is  removed.  The  fatty  acids  hereby  combine 
with  the  alkali,  forming  soaps,  while  the  neutral  fats  are  not  saponified 
under  these  conditions.  If  this  mixture  of  soaps  and  neutral  fats  is. 
treated  with  water  and  then  shaken  with  pure  ether,  the  neutral  fats 
are  dissolved,  while  the  soaps  remain  in  the  watery  solution.  The  fatty 
acids  may  be  separated  from  this  solution  by  the  addition  of  a  mineral 
acid  which  sets  the  acid  free. 

The  neutral  fats  separated  from  the  soaps  by  means  of  ether  are  often 
contaminated  with  cholesterin,  which  must  be  separated  in  quantitative 
determinations  by  saponification  with  alcoholic  caustic  potash.  The 
cholesterin  is  not  attacked  by  the  caustic  alkali,  while  the  neutral  fats  are 
saponified.  After  the  evaporation  of  the  alcohol  the  residue  is  dissolved  in 
water  and  shaken  with  ether,  which  dissolves  the  cholesterin.  The  fatty 
acids  are  separated  from  the  wratery  solution  of  the  soaps  by  the  addition  of 
a  mineral  acid.  If  a  mixture  of  soaps,  neutral  fats,  and  fatty  acids  is 
originally  present,  it  is  treated  first  with  water,  then  agitated  with  ether 
free  from  alcohol,  which  dissolves  the  fat  and  fatty  acids,  while  the  soaps 
remain  in  the  solution,  with  the  exception  of  a  very  small  amount  which  is 
dissolved  by  the  ether. 

To  detect  and  to  separate  the  different  varieties  of  neutral  fats  from 
each  other  it  is  best  first  to  saponify  them  with  alcoholic  potash,  or  still 
better  with  sodium  alcoholate,  according  to  Kossel,  Obermuller,  and 
Kruger.1  After  the  evaporation  of  the  alcohol  they  are  dissolved  in  water 
and  precipitated  with  sugar  of  lead.  The  lead  oleate  is  then  separated  from 
the  other  two  lead  salts  by  repeated  extraction  with  ether,  but  it  must  be 
remarked  that  the  lead  salts  of  the  other  fatty  acids  are  not  quite  insoluble 
in  ether.  The  residue  insoluble  in  ether  is  decomposed  on  the  water-bath 
with  an  excess  of  soda  solution,  evaporated  to  dyness,  finely  pulverized, 
and  extracted  with  boiling  alcohol.  The  alcoholic  solution  is  then  frac- 
tionally precipitated  by  barium  acetate  or  barium  chloride.  In  one  fraction 
the  amount  of  barium  is  determined,  and  in  the  other  the  melting-point  of 
the  fatty  acid  set  free  by  a  mineral  acid.  The  fatty  acids  occurring 
originally  in  the  animal  tissues  or  fluids  as  free  acids  or  as  soaps  are  con- 
verted into  barium  salts  and  investigated  as  above.  According  to  Jaeckle,2 
it  is  better  to  isolate  the  fatty  acids  as  silver  salts.  This  same  experimenter 
also  considers  it  more  advisable  to  dissolve  the  lead  salts  in  warm  benzene, 
as  suggested  by  Farnsteiner,  and  to  obtain  the  crystalline  lead  salts  of 
the  solid  fatty  acids  by  cooling. 

In  addition  to  the  methods  already  suggested  there  are  other  chemical  meth- 
ods which  are  important  in  investigating  fats.  Besides  ascertaining  the  melting- 
and  congealing-point  we  also  determine  the  following:  1.  The  acid  equivalent, 
which  is  a  measure  of  the  amount  of  fatty  acids  in  a  fat  and  is  determined  by 
titrating  the  fat  dissolved  in  alcohol-ether  with  N/10  alcoholic  caustic  potash,  using 


1  Zeitschr.  f.  physiol.  Chem.,  14,  15,  and  16. 
3  Ibid..  36. 


114  THE  ANIMAL  FATS. 

phenolphthalein  as  indicator.  2.  The  saponification  equivalent,  which  gives 
the  milligrams  of  caustic  potash  united  with  the  fatty  acids  in  the  saponification 
of  1  grm.  fat  with  N/2  alcoholic  caustic  potash.  3.  Reichert-Meissl's  equiva- 
lent, which  gives  the  quantity  of  volatile  fatty  acids  contained  in  a  given  amount 
of  neutral  fat  (5  grms.).  The  fat  is  saponified,  then  acidified  with  mineral  acid 
and  distilled,  whereby  the  volatile  fatty  acids  pass  over  and  the  distillate  is  ti- 
trated with  alkali.  4.  Iodine  equivalent  is  the  quantity  of  iodine  absorbed  by  a 
certain  amount  of  the  fat  by  addition.  It  is  chiefly  a  measure  of  the  quantity  of 
unsaturated  fatty  acids,  principally  oleic  acid  or  olein  in  the  fat.  Other  bodies, 
such  as  cholesterin,  may  also  absorb  iodine  or  halogens.  The  iodine  equiva- 
lent is  generally  determined  according  to  the  method  suggested  by  v.  Hubl. 
5.  The  acetyl  equivalent.  Oxyacids,  alcohols  such  as  cetyl  alcohol  or  cholesterin, 
and  those  constituents  of  fats  containing  the  OH  group  are  transformed  into  the 
corresponding  acetyl  ester  on  boiling  with  acetic  anhydride,  while  the  fatty  acids 
remain  unchanged,  and  in  this  way  the  estimation  of  these  bodies  is  possible.  The 
fat  is  saponified,  the  soaps  decomposed  by  an  excess  of  acid,  and  the  mixture 
of  fatty  acids,  oxyfatty  acids,  cholesterin,  etc.,  boiled  with  acetic  anhydride. 
The  acid  equivalent  is  determined  in  a  weighed  part  of  the  carefully  washed 
acetic-acid-free  mixture  by  titration  with  alcoholic  caustic  potash.  This  acid 
equivalent  represents  all  the  acids  (fatty  acids  as  well  as  the  acetylated 
oxyacids),  and  it  is  designated  the  acetyl  acid  equivalent.  The  neutral  fluid  is 
now  titrated  with  an  exactly  measured,  sufficient  quantity  of  the  same  alkali 
and  the  acetyl  compounds  saponified  by  boiling.  On  retitrating  we  find  the 
quantity  of  alkali  used  in  saponification,  and  this  number,  calculated  to  100  parts 
of  the  fat,  represents  the  acetyl  equivalent.  In  regard  to  the  performance  of 
the  above-mentioned  different  estimations  we  must  refer  the  reader  to  more  com- 
plete works,  such  as  "Analysis  of  Fats  and  Waxes,"  R.  Benedikt,  1897. 

In  the  quantitative  estimation  of  fats  the  finely  divided  dried  tissues  or 
the  finely  divided  residue  from  an  evaporated  fluid  is  extracted  with  ether, 
alcohol-ether,  benzene,  or  any  other  proper  extraction  medium.  The  investi- 
gations of  Dormeyer  1  and  others,  carried  on  in  Pfluger  's  laboratory, 
have  shown  that  even  with  very  prolonged  extraction  with  ether  all  the  fat 
is  not  extracted.  First  extract  the  greater  part  of  the  fat  by  ether.  Then 
digest  with  pepsin-hydrochloric  acid,  collect  the  insoluble  residue  on  a  filter, 
dry,  and  extract  with  ether.  The  fat  is  extracted  from  the  filtrate  by 
shaking  with  ether,  evaporating  the  extract  and  the  fat  separated  from  other 
bodies  by  extracting  the  residue  with  petroleum  ether.  Glikin,2  who  has 
tested  the  various  methods,  recommends  as  the  best  the  extraction  with 
boiling  petroleum  ether  and  the  removal  of  the  lecithin  by  acetone,  in 
which  it  is  insoluble. 

The  fats  are  poor  in  oxygen,  but  rich  in  carbon  and  hydrogen.  They 
therefore  represent  a  large  amount  of  chemical  potential  energy,  and  yield 
correspondingly  large  quantities  of  heat  on  combustion.  They  take  first 
rank  amongst  the  foods  in  this  regard,  and  are  therefore  of  very  great, 
importance  in  animal  life.     We  will  speak  more  in  detail  of  this  signifi- 

1  On  fat  extraction  for  quantitative  estimation  see:  Dormeyer,  Pfliiger's  Arch.,  61 
and  65;  Bogdanow,  ibid.,  65,  68,  and  Du  Bois-Reymond's  Arch.,  1897,  149;  N.  Schulz, 
Pfliiger's  Arch.,  66;  Voit  and  Krummacher,  Zeitschr.  f.  Biologie,  35;  0.  Frank,  ibid., 
35;  Polimanti,  Pfliiger's  Arch.,  70;  J.  Nerking,  ibid.,  71. 

2  Pfliiger's  Arch.,  95. 


SPERMACETI,  ETHAL,   AND  BEESWAX.  115 

cancc,  also  of  fat  format  ion  and  the  behavior  of  the  fata  in  the  body  in  the 
following  chapters. 

The  LECITHINS,  which  stand  in  close  relationship  to  the  fats,  will  be 
treated  in  a  subsequent  chapter.  The  following  bodies  are  related  to  the 
ordinary  animal  fats. 

Spermaceti.  In  the  living  spermaceti  or  white  whale  there  is  found  in  a  large 
cavity  in  the  skull  an  oily  liquid  called  spermaceti,  which  on  cooling  after  death 
separates  into  a  solid  crystalline  part,  ordinarily  called  SPERMACETI,  and  into  a 
liquid,  BPEBMACETI-OIL.  This  last  is  separated  by  pressure.  Spermaceti  is  also 
found  in  other  whales  and  in  certain  species  of  dolphin. 

The  purified,  solid  spermaceti,  which  is  called  CETIN,  is  a  mixture  of  esters  of 
fatty  aeids.  The  chief  constituent  is  the  eetyl-palmitic  ester  mixed  with  small 
quantities  of  compound  ethers  of  lauric,  myristic,  and  stearic  acids  with  radicals 
of  the  alcohols,  LETHAL,  C12H25.OH,  METHAL,  CmH.2,,OH,  and  BTETHAL,  C,JI  r.<dl. 

Cetin  is  a  snow-white  mass  shining  like  mother-of-pearl,  crystallizing  in  pi 
brittle,  fatty  to  the  touch,  and  which  has  a  varying  melting-point  of  30°  to 
50°  C,  depending  upon  its  purity.  Cetin  is  insoluble  in  water,  but  dissolves 
easily  in  cold  ether  or  volatile  and  fatty  oils.  It  dissolves  in  boiling  alcohol, 
but  crystallizes  on  cooling.  It  is  saponified  with  difficulty  by  a  solution  of  caustic 
] iot ash  in  water,  but  with  an  alcoholic  solution  it  saponifies  readily  and  the  above- 
mentioned  alcohols  are  set  free. 

CH3 

Ethal  or  cetyl  alcohol,  C18H340  =  (CH2)U,  which  also  occurs  in  the  coccygeal 

CH2.OH 
gland  of  ducks  and  geese  (De  Jonge  x)   and  in  smaller  quantities  in  beeswax, 
and  found  by  Ludwig  and  v.  Zeynek  2  in  the  fat  from  dermoid  cysts,  forms  white, 
transparent,  odorless,  and  tasteless  crystals  which  are  insoluble  in   water  but 
dissolve  easily  in  alcohol  and  ether.     Ethal  melts  at  49.5°  C. 

Spermaceti-oil  yields  on  saponification  valerianic  acid,  small  amounts  of 
solid  fatty  acids,  and  physetoleic  acid.  This  acid,  which  has,  like  hypogaeic 
acid,  the  composition  C,„H30O2,  occurs  also,  as  found  by  Ljtjbarsky,3  in  con- 
siderable amounts  in  the  fat  of  the  seal.  It  forms  colorless  and  odorless,  needle- 
shaped  crystals  which  easily  dissolve  in  alcohol  and  ether  and  melt  at  34°  C. 

Beeswax  may  be  treated  here  as  concluding  the  subject  of  fats.  It  con- 
tains three  chief  constituents:  (1)  Cerotic  acid,  C27H5202,4  which  occurs  as  cetyl 
ether  in  Chinese  wax  and  as  free  acid  in  ordinary  wax.  It  dissolves  in  boiling 
alcohol  and  separates  as  crystals  on  cooling.  The  cooled  alcoholic  extract  of 
wax  contains  (2)  cerolein,  which  is  probably  a  mixture  of  several  bodies,  and 
(3)  myricin,  which  forms  the  chief  constituent  of  that  part  of  wax  which  is  in- 
soluble in  warm  or  cold  alcohol.  Myricin  consists  chiefly  of  palmitic-acid  ether 
of  melissyl  (myricyl)  alcohol,  C^H^/OH.  This  alcohol  is  a  silky,  shining,  crys- 
talline body  melting  at  85°  C. 

1  Zeitschr.  f.  physiol.  Chem.,  3. 
2Ibid.,2Z. 

3  Journ.  f.  prakt.  Chem.  (N.  F.),  57. 

4  See  Henriques,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30,  1415. 


CHAPTER  V. 
THE  ANIMAL  CELL. 

The  cell  is  the  unit  of  the  manifold,  variable  forms  of  the  organism;  it 
forms  the  simplest  physiological  apparatus,  and  as  such  is  the  seat  of  chem- 
ical processes.  It  is  generally  admitted  that  all  chemical  processes  of 
importance  do  not  take  place  in  the  animal  fluids,  but  transpire  in  the 
cells,  which  may  be  considered  as  the  chemical  laboratory  of  the  organism. 
It  is  also  principally  the  cells  which,  through  their  greater  or  less  activity, 
regulate  or  govern  the  range  of  the  chemical  processes  and  also  the 
intensity  of  the  total  exchange  of  material. 

It  is  natural  that  the  chemical  investigation  of  the  animal  cell  should 
in  most  cases  coincide  with  the  study  of  those  tissues  of  which  it  forms  the 
chief  constituent.  Only  in  a  few  cases  can  the  cells,  by  relatively  simple 
manipulations,  be  directly  isolated  in  a  rather  pure  state  from  the  tissues, 
as,  for  example,  in  the  investigation  of  pus  or  of  tissues  very  rich  in  cells. 
But  even  in  these  cases  the  chemical  investigation  may  not  lead  to  any 
positive  results  in  regard  to  the  constituents  of  the  uninjured  living  cells. 
By  the  process  of  chemical  transformation  new  substances  may  be  formed 
on  the  death  of  the  cell,  and  at  the  same  time  physiological  constituents 
of  the  cell  may  be  destroyed  or  transported  into  the  surrounding  medium 
&nd  therefore  escape  investigation.  For  this  and  other  reasons  we  possess 
only  a  very  limited  knowledge  of  the  constituents  and  the  composition 
of  the  cell,  especially  of  the  living  one. 

"While  young  cells  of  different  origin  in  the  early  period  of  their  exist- 
ence may  show  a  certain  similarity  in  regard  to  form  and  chemical  com- 
position, they  may,  on  further  development,  not  only  take  the  most  varied 
forms,  but  may  also  offer  from  a  chemical  standpoint  the  greatest  diversity. 
As  a  description  of  the  constituents  and  composition  of  the  different  cells 
occurring  in  the  animal  organism  is  nearly  equivalent  to  a  demonstration 
of  the  chemical  properties  of  most  animal  tissues,  and  as  this  exposition 
will  be  found  in  the  corresponding  chapters,  we  will  here  only  discuss  the 
chemical  constituents  of  the  young  cells  or  cells  in  general. 

In  the  study  of  these  constituents  we  are  confronted  with  another 
difficulty,  namely,  we  must  differentiate  by  chemical  research  between 
those  constituents  which  are  essentially  necessary  for  the  life  of  the  cells 

116 


CELL  PROTOPLASM.  117 

and  those  which  are  casual,  i.e.,  stored  up  as  reserve  material  or  as  meta- 
bolic products.  In  this  connection  we  have  only  been  able,  thus  far,  to 
learn  of  certain  substances  which  seem  to  occur  in  every  developing  cell. 
Such  bodies,  called  PRIMARY  by  KOSSEL,1  arc,  besides  water  and  certain 
mineral  constituents,  proteids,  nucleoproteida  or  QUcleins,  lecithins, 
glycogen  (?),  and  cholesterin.  Those  bodies  which  do  not  occur  in  every 
developing  poll  are  called  skcuxdahv.  Amongst  these  we  have  fat, 
glycogen  (?),  pigments^eje.  It  must  not  be  forgotten  that  it  is  still  possible 
that  other  primary  cell  constituents  may  exist,  but  unknown  to  us,  and 
we  also  do  not  know  whether  all  the  primary  constituents  of  the  cell  are 
necessary  or  essential  for  the  life  and  functions  of  the  same. 

Another  important  question  is  the  division  of  the  various  cell  constit- 
uents between  the  two  morphological  components  of  the  cell,  namely,  the 
protoplasm  and  the  nucleus.  This  is  very  difficult  to  decide  for  many  of 
the  constituents;  nevertheless  it  is  appropriate  to  differentiate  between  the 
protoplasm   and  the  nucleus. 

The  Protoplasm  of  the  developing  cell  consists  during  life  of  a  semi- 
solid mass,  contractile  under  certain  conditions  and  readily  changeable, 
which  is  rich  in  water  and  whose  chief  portion  consists  of  protein  substances, 
i.e.,  of  colloids.  If  the  cell  be  deprived  of  the  physiological  conditions  of 
life,  or  if  exposed  to  destructive  exterior  influences,  such  as  the  action  of 
high  temperatures,  of  chemical  agents,  the  protoplasm  dies.  The  proteid 
bodies  which  it  contains  coagulate  at  least  partially,  and  other  chemical 
changes  are  found  to  take  place.  The  alkaline  reaction  (litmus)  of  the 
living  cell  may  become  acid  by  the  appearance  of  parabiotic  acid,  and  the 
carbohydrate,  glycogen,  which  habitually  occurs  in  the  young  growing  cell, 
may  after  its  death  be  quickly  changed  and  consumed. 

The  questionas_to  the  internal  structure  of  the  protoplasm  is  still  ir^ 
controversy.  It  is  of  little  importance  in  the  study  of  the  chemical  compo- 
sition of  the  cells,  as  it  is  impossible  to  study,  especially  by  chemical  means, 
the  morphologically  different  constituents  of  the  protoplasm.  With  the 
exception  of  a  few  microchemical  reactions  the  chemical  analysis  has  been 
restricted  to  the  protoplasm  as  such,  and  the  investigations  have  been 
directed  in  the  first  place  to  the  protein  substances  which  form  the  chief 
mass  of  the  protoplasm. 

The  proteids  of  the  protoplasm  consist,  according  to  the  older  general 
view,  chiefly  of  globulins.  Albumins  have  also  been  found  besides  the  globu- 
lins. There  is  no  doubt  at  present  that  the  albumins  occur  in  the  celLs  only 
as  traces,  or  at  least  only  in  trifling  quantities.  The  presence  of  globulins 
can  hardly  be  disputed,  although  certain  cell  constituents  described  as 
globulins  have  been  shown  on  closer  investigation  to  be  nucleoalbumins  or 

1  Verhandl.  d.  physiol.  Gesellsch.  zu  Berlin,  1S90-91,  Nos.  5  and  6. 


118  THE  ANIMAL  CELL. 

nucleoproteids.  According  to  Halliburton  x  the  proteid  occurring  in  all 
cells  and  coagulating  at  47°-50°  C.  is  a  true  globulin. 

In  opposition  to  the  view  that  the  chief  mass  of  the  animal  cell  consists 
of  true  proteids,  Hammarsten  2  expressed  the  opinion  several  years  ago  that 
the  chief  mass  of  the  protein  substances  of  the  cells  does  not  consist  of 
proteids  in  the  ordinary  sense,  but  consists  of  more  complex  phosphorized 
bodies,  and  that  the  globulins  and  albumins  are  to  be  considered  as  nutri- 
tive material  for  the  cells  or  as  destructive  products  in  the  chemical  trans- 
formation  of  the  protoplasm.  This  view  has  received  substantial  support 
by  investigations  within  the  last  few  years.  Alex.  Schmidt  3  has  come  to 
the  view,  by  investigations  on  various  kinds  of  cells,  that  they  contain 
only  very  little  proteid,  and  that  the  chief  mass  consists  of  very  complex 
protein   substances. 

The  protein  substances  of  the  cells  consist  chiefly  of  compound  proteids, 
and  these  are  divided  between  the  glucoproteid  and  the  nucleoproteid 
groups.  It  is  impossible  at  present  to  state  to  what  extent  nucleoalbumins 
exist  in  the  cells  because  thus  far  in  most  cases  no  exact  difference  has  been 
made  between  them  and  the  nucleoproteids.  Hoppe-Seyler  4  calls  vitellin 
a  regular  constituent  of  all  protoplasm.  This  body  used  to  be  considered 
as  a  globulin,  but  later  researches  have  shown  that  the  so-called  vitellin 
bodies  may  be  of  various  kinds.  Certain  vitellins  seem  to  be  nucleoalbu- 
mins, and  it  is  therefore  very  probable  that  cells  habitually  contain  nucleo- 
albumins. 

The  nucleoproteids  take  a  very  prominent  place  among  the  compound 
proteids  of  the  cell.  The  various  substances  isolated  by  different  investiga- 
tors from  animal  cells,  such  as  tissue-fibririQggn  (Wooldridge),  cytoglobin 
and  prdglobulin  (Alex.  Schmidt),  or  nucleohiston  (Kossel  and  Lilien- 
feld  5),  belong  to  this  group.  The  cell  constituent  which  swells  up  to  a 
sticky  mass  with  common  salt  solution  and  called  Rovida's  hyaline  sub- 
stance also  belongs  to  this  group. 

The  above-mentioned  different  protein  substances  have  only  been  simply 
designated  as  constituents  of  the  cells.  The  next  question  is  which  of  these 
belong  to  the  protoplasm  and  which  to  the  nucleus.  At  present  we  can 
give  no  positive  answer  to  this  question.  According  to  Kossel  and  Lilien- 
feld,6  the  cell-nucleus  of  the  leucocytes  of  the  thymus  gland  contains  a 

1  See  Halliburton,  On  the  Chemical  Physiology  of  the  Animal  Cell,  1893,  No.  1, 
King's  College  Physiol.  Laboratory. 
2Pfluger's  Arch.,  30. 

3  Alex.  Schmidt,  Zur  Blutlehre.     Leipzig,  1892. 

4  Physiol.  Chem.,  1877-1881,  76. 

5  See  L.  C.  Wooldridge,  Die  Gerinnung  des  Blutes.  Leipzig,  1891 ;  A.  Schmidt, 
Zur  Blutlehre;   Lilienfeld,  Zeitschr.  f.  physiol.  Chem.,  18. 

c  Ueber  dio  Wahlverwandtschaft  der  Zellelemente  zu  gewissen  Farbstoffen  Ver- 
handl.  d.  physiol.  Gesellsch.  zu  Berlin,  No.  11,1893. 


PROTEINS  OF   THE  CELL.  119 

nucleoproteid  as  chief  constituent,  besides  nucleins,  and  sometimes  perhaps 
also  nucleic  acid  (see  below),  while  the  body  of  the  cells  contains  chiefly 
pure  proteida  besides  other  substances,  and  only  a  nucleoproteid,  con- 
taining a  very  small  quantity  of  phosphorus.  As  the  lymphocytes  of  the 
thymus  gland  of  the  calf  contain  only  one  nucleus,  in  which  the  mac 
the  nucleus  surpasses  that  of  the  cytoplasm,  it  is  natural  that  the  relative 
proportion  of  the  various  protein  substances  in  these  cells  cannot  be  taken 
as  a  standard  for  the  composition  of  other  cells  richer  in  cytoplasm. 

Complete  investigations  in  regard  to  the  distribution  of  protein  sub- 
stances in  the  protoplasm  and  nucleus  of  other  cells  have  not  been  made. 
If  we  consider  for  the  present  that  the  cells  rich  in  protoplasm  contain,  as 
a  ride,  only  very  little  true  proteid,  we  are  hardly  wrong  in  considering  it 
probable  that  the  protoplasm  contains  chiefly  nucleoalbumins  and  compound 
proteids  besides  traces  of  albumin  and  a  little  globulin.  These  compound 
proteids  are  in  certain  cases  glucoproteids,  but  otherwise  nucleoproteids, 
which  differ  from  the  nucleoproteids  of  the  nucleus  in  being  poorer  in 
phosphorus,  besides  containing  a  great  deal  of  proteid  and  only  less  of  the 
prostetic  group,  and  hence  have  no  specially  pronounced  acid  character. 

The  nucleoproteids  of  the  nucleus  are  on  the  contrary,  as  showji  by 
Lilienfeld  and  Kossel,  rich  in  phosphorus  and  of  a  strongly  acid  charac- 
ter. These  nucleoproteids  will  be  treated  in  speaking  of  the  nucleic  acids 
of  the  nucleus. 

In  cases  in  which  the  protoplasm  is  surrounded  by  an  outer,  condensed 
layer  or  a  cell  membrane,  this  envelope  seems  to  consist  of  albumoid  sub- 
stances. In  a  few  cases  these  substances  seem  to  be  closely  related  to 
elastin;  in  other  cases,  on  the  contrary,  they  seem  rather  to  belong  to  the 
keratin  group.  Even  in  cells  which  do  not  seem  to  have  any  visible  special 
layers  forming  boundaries,  we  still  admit  of  such  layers  on  account  of  the 
behavior  of  the  cells  as  regards  permeability. 

Nernst  *  has  shown  by  a  special  experiment  that  the  permeability  of  a 
membrane  for  a  certain  substance  is  essentially  dependent  upon  the  sol- 
vent power  of  the  membrane  for  the  said  substance.  This  point,  which 
is  of  the  greatest  importance  in  the  stud}'  of  osmotic  phenomena  in  living 
cells,  has  been  specially  investigated  by  Overton.2  The  behavior  of  the 
living  cells  towards  dyestuffs,  also  the  ready  introduction  into  animal 
and  plant  protoplasm  of  such  bodies  as  are  insoluble  or  only  slightly 
soluble  in  water  but  readily  soluble  in  fats  or  fat-like  bodies  has  led 
Overton  to  conclude  that  the  protoplasm-boundary  layer  behaves  like  a 
substance  layer  whose  solvent  power  is  closely  related  to  the  fatty  oils. 
According  to  this  investigator,  the  protoplasm-boundary  layer  is  probably 

1  Zeitschr.  f.  physikal  Chem.,  6. 

5  Yierteljahrssehr.  d.  Naturf.  Ges.  in  Zurich,  44  (1S99),  and  Overton,  Studien  iiber 
die  Xarkose,  Jena,  1901. 


120  THE  ANIMAL  CELL. 

impregnated  with  lipoids,  i.e.,  with  lecithins,  cholesterin,  and  bodies  sinulai 
to  protagon,  and  among  which  lecithin,  which  also  takes  up  water,  must 
be  of  the  greatest  importance. 

The  cholesterins  and  the  protagons  will  be  best  treated  in  another 
connection  (see  Chapters  VIII  and  XII).  We  will  only  discuss  here  the 
lecithin  which  is  present  in  every  cell. 

Lecithins.  These  bodies  are  ester  compounds  *  of  glycerophosphoric  acid 
substituted  by  two  fatty-acid  radicals,  with  a„  base  called  choline.  Accord- 
ing to  the  kind  of  fatty  acid  contained  in  the  lecithin  molecule  it  is  possible 
to  have  various  lecithins,  such  as  stearyl-,  palmityl-,  and  oleyl-lecithins. 
According  to  Thudichum  2  two  different  fatty  acids  may  exist  simultane- 
ously in  one  lecithin,  and  according  to  him  every  true  lecithin  always  con- 
tains at  least  one  oleic-acid  radical.3  All  lecithins  are  mono-nitrogenous 
mono-phosphatides,  which  contain  1  atom  of  nitrogen  for  every  atom  of 
phosphorus.  As  an  example  of  a  lecithin  we  give  the  one  closely  studied 
by  Hoppe-Seyler  and  Diaconow,4  called  distearyl-lecithin,  C44H80NPOa= 

CH2 — 0 — C18H350 

CH  -0- C18H350 

CH2— 0\ 

HO7P0. 

/C2H-0/ 

nAch3)3 

On  saponification  with  alkalies  or  baryta-water  lecithin  yields  fatty 
acids,  glycerophosphoric  acid,  and  choline.  It  is  only  slowly  decomposed 
by  dilute  acids.  Besides  small  quantities  of  glycerophosphoric  acid  (per- 
haps also  distearylglycerophosphoric  acid)  we  have  large  quantities  of  free 

phosphoric  acid  split  off. 

CH2.OH 
I 
Glycerophosphoric  acid,  C3H9P06  =  CH.OH  ,  is  a  bibasic  acid  which  proba- 

CH—  Ox 
OH-^PO 
OH/ 
bly  only  occurs  in  the  animal  fluids  and  tissues  as  a  cleavage  product  of  lecithins. 

/CH2.CH,(OH) 
^lf-(CHN 
\OH 

which  occurs  extensively  in  the  plant  kingdom,  is  not  identical  with  the  base, 
Neurin,  prepared    by  Liebreich  as  a  decomposition  product  from  the  brain, 

1  Strecker,  Annal.  d.  Chem.  u.  Pharm.,  148;  Hundeshagen,  Journ.  f.  prakt.  Chem. 
(N.  F.),  28;  Gilson,  Zeitschr.  f.  physiol.  Chem.,  12. 

2  J.  L.  W.  Thudichum,  Die  chemische  Konstitution  des  Gehirns  des  Menschen,  etc. 
Tubingen,  1901. 

8 See  Henriques  and  Hansen,  Skan.  Arch.  f.  Physiol.,  14. 
4  Hoppe-Seyler,  Med.  chem.  Untersuch.,  Fleft  2  and  3. 


Choline  (trimethyloxyethylammonium  hydroxide),  C5H,5N02  =N—  (CH3)3, 


LECITHINS.  121 

which  is  considered  as  brimethylvmylammonium  hxdroxide,  CLHuNO.  Choline 
is  ;i  sirupv  fluid  readily  miscible  with  absolute  alcohol.  Hydrochloric  acid  gives  a 
combination  which  is  very  soluble  in  water  and  alcohol,  bul  insoluble  in  ether, 
chloroform,  and  benzene.  This  compound  forms  a  double  combination  with  plati- 
num chloride  which  is  soluble  in  water,  insoluble  in  absolute  alcohol  and  ether, 
crystallizing  ordinarily  in   six-sided  orange-colored   plates.      This  combination  is 

used  in  the  detection  and  identification  of  this  base.  Choline  also  forms  a  crys- 
talline double  combination  with  mercuric  chloride  and  with  gold  chloride.1  On 
heating  the   free  base  it   decomposes  into  trimethylamine,  ethylene  oxide,  and 

water. 

Lecithin  occurs,  as  Hoppe-Seyler  2  has  especially  shown,  widely  diffused 
in  thevegetable  and  animal  kingdoms.  According  to  this  investigator,  it 
occurs  also  in  many  cases  in  loose  combination  with  other  bodies,  such  as 
proteids,  haemoglobin,  and  others.  Lecithin,  according  to  Hoppe-Seyler, 
is  found  in  nearly  all  animal  and  vegetable  cells  thus  far  studied,  and  also 
in  nearly  all  animal  fluids.  It  is  especially  abundant  in  the  brain,  nerves, 
fish-eggs,  yolk  of  the  egg,  electrical  organs  of  the  Torpedo  electricus,  semen, 
and  pus,  and  also  in  the  muscles  and  blood-corpuscles,  blood-plasma,  lymph, 
milk,  especially  woman's  milk,  and  bile.  Lecithin  is  also  found  in  differ- 
ent pathological  tissues  or  liquids. 

This  wide  distribution  of  the  lecithins,  as  also  the  fact  that  it  is  a 
primary  cell  constituent,  gives  great  physiological  importance  to  these 
substances.  "We  have  in  lecithin,  no  doubt,  a  very  important  material  for 
the  building  up  of  the  complicated  phosphorized  nuclein  substances  of  the 
cell  and  cell  nucleus.  That  the  lecithins  are  of  great  importance  in  the 
development  and  growth  of  living  organisms,  in  fact  for  the  bioplastic 
processes  in  general,  follows  also  from  several  investigations.3 

Lecithin  may  be  obtained  in  grains  or  warty  masses  composed  of  small 
crystalline  plates  by  strongly  cooling  its  solution  in  strong  alcohol.  In  the 
dry  state  it  has  a  waxy  appearance,  is  plastic  and  soluble  in  alcohol,  espe- 
cially on  heating  (to  40-50°  C.) ;  it  is  less  soluble  in  ether.  It  is  dissolved 
also  by  chloroform,  carbon  disulphide,  benzene,  and  fatty  oils.  The  solu- 
tion of  lecithin  in  alcohol-ether  or  chloroform  is  precipitated  by  acetone. 
It  swells  in  water  to  a  pasty  mass  which  shows  under  the  microscope  slimy, 
oily  drops  and  threads,  so-called  myelin  forms  (see  Chapter  XII).  On 
warming  this  swollen  mass  or  the  concentrated  alcoholic  solution,  decom- 
position takes  place  with  the  production  of  a  browm  color.  On  allowing  the 
solution  or  the  swollen  mass  to  stand,  decomposition  takes  place  and  the 
reaction  becomes  acid. 

1  In  regard  to  choline  and  its  compounds  see  Gulewitsch,  Zeitschr.  f.  physiol. 
Chem.,  24. 

2  Physiol.  Chemie.     Berlin,  1877-1881,  57. 

'See  Stoklasa,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29;  Wiener  Sitzungsber. ,  104; 
Zeitschr.  f.  phvsiol.  Chem.,  25;  and  W.  Danielewsky,  Comp.  rend.,  121  and  123,  and 
W.  Koch,  Zeitschr.  f.  physiol.  Chem.,  37. 


122  THE  ANIMAL  CELL. 

With  considerable  water,  lecithin  gives  an  emulsion  or  indeed  a  filter- 
able colloidal  solution,  which  is  precipitated  by  salts  with  divalent  cations, 
such  as  Ca,  Mg,  and  others  (W.  Koch).  This  precipitate  dissolves  again 
in  water  after  the  removal  from  the  solution  of  the  electrolytes,  and  the 
formation  of  this  precipitate  can  be  prevented  by  the  presence  of  salts  of 
monovalent  cations. 

We  are  here  not  dealing  with  a  chemical  but  rather  a  physical  pre- 
cipitation reaction  (Koch  x).  In  putrefaction  lecithin  yields  glycerophos- 
phoric  acid  and  choline;  the  latter  further  decomposes  with  the  forma- 
tion of  methylamine,  ammonia,  carbon  dioxide,  and  marsh-gas  (Hase- 
broek  2).  If  dry  lecithin  be  heated  it  decomposes,  takes  fire  and  burns, 
leaving  a  phosphorized  ash.  On  fusing  with  caustic  alkali  and  saltpeter 
it  yields  alkali  phosphates.  Lecithin  is  easily  carried  down  during  the 
precipitation  of  other  compounds  such  as  the  proteid  bodies,  and  may 
therefore  very  greatly  change  the  solubilities  of  the  latter. 

Lecithin  combines  with  acids  and  bases.  The  combination  with  hydro- 
chloric acid  gives  with  platinum  chloride  a  double  salt  which  is  insoluble 
in  alcohol,  soluble  in  ether,  and  which  contains  10.2  per  cent  platinum 
(for  distearyllecithin) .  The  cadmium-chloride  compound  which  contains  3 
molecules  of  lecithin  and  4  molecules  of  cadmium  chloride  (Ulpiani  3) 
is  difficultly  soluble  in  alcohol,  but  dissolves  in  a  mixture  of  carbon  disul- 
phide  and  ether  or  alcohol. 

It  may  be  prepared  tolerably  pure  from  the  yolk  of  the  hen's  egg  by  the 
following  methods,  as  suggested  by  Hoppe-Seyler  and  Diaconow.  The 
yolk,  deprived  of  proteid,  is  extracted  with  cold  ether  until  all  the  yellow 
color  is  removed.  Then  the  residue  is  extracted  with  alcohol  at  50-60°  C. 
After  the  evaporation  of  the  alcoholic  extract  at  50-60°  C,  the  sirupy 
matter  is  treated  with  ether  and  the  insoluble  residue  dissolved  in  as  little 
alcohol  as  possible.  On  cooling  this  filtered  alcoholic  solution  to  —5°  to 
— 10°  C.  the  lecithin  gradually  separates  in  small  granules.  The  ether, 
however,  contains  considerable  of  the  lecithin.  The  ether  is  distilled  off 
and  the  residue  dissolved  in  chloroform  and  the  lecithin  precipitated  from 
this  solution  by  means  of  acetone  (Altmann). 

According  to  Gilson,4  a  new  portion  of  lecithin  may  be  obtained  from 
the  ether  used  in  extracting  the  yolk  by  dissolving  the  residue  after  the 
evaporation  of  the  ether  in  petroleum  ether  and  then  shaking  this  solu- 
tion with  alcohol.  The  petroleum-ether  takes  the  fat,  while  the  lecithin 
remains  dissolved  in  the  alcohol  and  may  be  obtained  therefrom  rather 
easily  by  using  the  proper  precautions,  as  described  in  the  original  publi- 
cation. 

1  Zeitschr.  f.  physiol.  Chem.,  37. 

2  Ibid.,  12. 

•  Chem.  Centralbl.,  1901,  II,  SO  and  193. 

4  Altmann,  cited  from  Hoppe-Seyler-Thierf elder's  Handbuch,  7.  Auflage;  Gilson, 
ibid. 


LLC  ITU  INS.  123 

Zuelzer's  method  is  based  upon  the  precipitability  of  the  lecithin  by 
acetone,  and  Bergell's1  method  upon  the  preparation  of  the  double 
salt  of  cadmium  and  its  decomposition  by  ammonium  carbonate.  The 
preparations  obtained  by  the  different  methods  consists  generally  of  a 
mixture  of  lecithins. 

The  detection  ami  the  quantitative  estimation  of  lecithin  in  animal 
fluids  or  tissues  is  based  on  the  solubility  of  the  lecithin  (at  50-60°  C.)  in 
alcohol-ether,  by  which  the  phosphoric  acid  or  glycerophosphoric  acid 
salts  which  may  be  present  at  the  same  time  are  not  dissolved.  The 
alcohol-ether  extract  is  evaporated,  the  residue  dried  and  fused  with  soda 
and  saltpeter.  Phosphoric  acid  is  formed  from  the  lecithin,  and  it  can  be 
used  in  the  detection  and  quantitative  estimation.  The  distearyllecithin 
yields  s.T'.ix  per  cent  P205.  This  method  is,  however,  not  exactly  correct, 
for  it  is  possible  that  other  phosphorized  organic  combinations,  such  as 
jecorin  (see  Chapter  Mil)  and  protagon  (Chapter  XII)  may  have  passed 
into  the  alcohol-ether  extract.  In  detecting  lecithin  the  double  combina- 
tion of  choline  and  platinum  must  also  be  prepared.  The  residue  of  the 
evaporated  alcohol-ether  extract  may  be  boiled  for  an  hour  with  baryta- 
water,  filtered,  the  excess  of  barium  precipitated  with  C02,  and  filtered 
while  hot.  The  filtrate  is  concentrated  to  a  sirupy  consistency,  extracted 
with  absolute  alcohol,  and  the  filtrate  precipitated  with  an  alcoholic  solu- 
tion of  platinum  chloride.  The  precipitate  after  filtration  may  be  dissolved 
in  water  and  allowed  to  crystallize  over  sulphuric  acid. 

Prolagons,  which  are  found  in  the  leucocytes  and  pus-cells,  are  also  to 
be  considered  as  a  constituent  of  protoplasm.  These  phosphorized  bodies 
occur  principally  in  the  brain  and  nerves,  and  hence  will  be  described  in  a 
following  chapter  (XII). 

Glycogen,  first  discovered  by  Cl.  Bernard,  is  found  in  developing 
animal  cells  and  especially  in  developing  embryonic  tissues.  According 
to  Hoppe-Seyler  it  seems  to  be  a  never-failing  constituent  of  the  cells 
which  show  amoeboid  movement,  and  he  found  this  carbohydrate  in  the 
leucocytes,  but  not  in  the  developed  motionless  pus-corpuscles.  Salomon 
and  afterwards  others  have,  however,  found  glycogen  in  pus.2  From  the 
relationship  which  seems  to  exist  between  glycogen  and  muscular  work  (see 
Chapter  XI),  it  is  presumable  that  a  consumption  of  glycogen  takes  place 
in  the  movement  of  animal  protoplasm.  On  the  other  hand,  the  extensive 
occurrence  of  glycogen  in  embryonic  tissues,  as  also  its  occurrence  in  patho- 
logical  tumors  and  in  abundant  cell  formation,  speaks  for  the  importance 
of  this  body  in  the  formation  and  development  of  the  cell. 

In  adult  animals  glycogen  occurs  as  stored  foodstuff  in  the  muscles  and 
certain  other  organs,  but  principally  in  the  liver;  therefore  it  will  be  com- 
pletely described  in  connection  with  this  organ  (Chapter  VIII). 

Another  body,  or  perhaps  more  correctly  a  group  of  bodies  which  occur 


1  Zuelzer,  Zeitschr.  f.  physiol.  Chem.,  27,  and  Bergell,  Ber.  d.  d.  chem.  Gesellsch.,  33. 
1  In  regard  to  the  literature  ou  glycogen  see  Chap.  VIII. 


124  THE  ANIMAL  CELL. 

widely  distributed  in  the  animal  and  vegetable  kingdoms,  and  which  are 
present  regularly  in  the  cells,  are  the  cholesterins.  The  best-known  repre- 
sentative of  this  group  is  ordinary  cholesterin  (see  Chapter  VIII),  which  is 
the  chief  constituent  of  certain  biliary  calculi  and  exists  in  abundant  quan- 
tities in  the  brain  and  nerves.  It  is  hardly  admissible  that  this  body  is  of 
direct  importance  for  the  life  and  development  of  the  cell.  It  must  be 
considered  that  the  cholesterin,  as  accepted  by  Hoppe-Seyler,1  is  a  cleavage 
product  appearing  in  the  cell  during  the  processes  of  life,  but  this  does  not 
exclude  the  possibility  that  the  cholesterin,  as  a  constituent  of  the  lipoids  of 
the  protoplasm-boundary  layers  (Overton),  may  be  of  indirect  importance 
in  cell-life.  According  to  Hoppe-Seyler  the  same  is  true  for  the  fats, 
which  do  not  occur  constantly  in  the  cells  and  have  nothing  to  do  in  the 
ordinary  processes  of  life.  There  is  no  doubt  that  cholesterin  exists  as  a 
constituent  of  the  protoplasm,  but  its  existence  in  the  nucleus  is  question- 
able. The  intracellular  enzymes  are  undoubtedly  constituents  of  the 
protoplasm  as  well  as  the  nucleus  and  must  be  of  the  greatest  importance 
for  the  life  and  functions  of  the  cells. 

The  cell  nucleus  has  a  rather  complex  structure.  It  consists  in  part  of 
fibriles  which  form  a  network  and  another  part  which  is  less  solid  and 
homoflpin^ous.  The  first  differs  from  the  second  in  possessing  a  stronger 
affinity  for  many  dyes.  On  account  of  this  behavior  the  first  is  called  the 
chromatic  substance  or  chromatin,  and  the  other  the  achromatic  substance 
or  achromatin. 

The  homogeneous  substance  of  the  nucleus  is  considered  as  a  mixture 
of  proteid.  The  network  seems  to  contain  the  more  specific  constituent 
of  the  nucleus,  namely,  the  nuclein  substances.  Besides  this  it  is  alleged 
to  contain  another  substance  also,  plastin.  This  last  is  less  soluble  than 
the  nuclein  substances  and  does  not  have  the  property,  like  them,  of  fixing 
dyes. 

1        The    chief    constituents    of    the    cell  nucleus    are    the  nucleoproteids 
and  in  certain  cases"  the  nucleic  acids. 

Nucleoproteids.  The  most  important  of  these  bodies  have  already 
been  discussed  in  a  previous  chapter  (II,  page  56).  These  bodies  are 
strong  or  loose  combinations  of  nucleic  acids  with  proteid.  To  the  latter 
belongs  histon  in  certain  cases,  and  the  compounds  between  nucleic  acids 
and  protamins  should  also  perhaps  be  called  nucleoproteids.  There  is  a 
difference  among  the  nucleoproteids  dependent  on  the  various  proteid 
complexes  as  well  as  upon  the  nucleic  acids.  They  contain  generally  con- 
siderable proteid  in  the  molecule,  hence  they  give  the  ordinary  proteid 
reactions,  and  therefore  are  closely  related  to  the  proteid  bodies.  .The 
nucloproteids  occurring  in  the  cell  nucleus  seem  to  be  characterized  by 


1  Physiol.  Chemie,  81. 


NUCLB1NS.  125 

containing  a  relative  large  amount  of  phosphorus  and  a  pronounced  arid 
character. 

In  the  preceding  discussion  of  the  nucleoproteids  attention  Avas  called  to 
the  fact  that,  OB  their  modification  by  heat,  by  weak  acid  action,  and  by 
peptic  digestion,  proteid  is  split  off  and  a  nucleoproteid  richer  in  phosphorus 
is  formed.  These  compound  proteids,  rich  in  nucleic  acid,  obtained  by- 
peptic  digestion  from  cells,  cell-rich  organs,  or  nucleoproteids  have  been 
called  nuclcin  (Miescher,  Hoppe-Seyler  ')  or  true  nucleins.  But  as  the 
true  nuclein  seems  to  be  nothing  but  a  modified  nucleoproteid  poor  in  pro- 
teid, it  seems  unnecessary  to  give  the  name  nuclein  thereto.  On  the  other 
hand,  the  nucleins  have  other  properties  than  the  nucleoproteids,  and  as 
the  nucleins  bear  the  same  relationship  to  the  nucleoproteids  that  the 
pseudonuclein  does  to  the  nucleoalbumins,  we  will  give  here  a  short  de- 
scription of  the  nucleins  as  well  as  the  pseudo-  or  para-nucleins. 

Nucleins  or  true  nucleins  are  formed,  as  above  stated,  from  nucleopro- 
teids in  their  peptic  digestion  or  by  treatment  with  dilute  acids.  It  must 
be  remarked  that  the  nucleins  are  not  entirely  resistant  towards  gastric 
juice  and  also  that  at  least  one  nucleoproteid,  namely,  the  one  obtained 
from  the  pancreas,  completely  dissolves,  leaving  no  nuclein  residue  on 
treatment  with  gastric  juice  (Umber,  Milroy  2).  The  nucleins  are  rich 
in  phosphorus  containing  in  the  neighborhood  of  5  per  cent.  According  to 
LlEBEEMANN8  metaphosphoric  acid  can  be  split  off  from  true  nucleins  (yeast 
nuclein).  The  nucleins  are  decomposed  into  proteid  and  nucleic  acid  by 
caustic  alkali,  and  as  different  nucleic  acids  exist,  so  also  there  exist  differ- 
ent nucleins.  As  previously  stated,  proteids  may  be  precipitated  in  acid 
solutions  by  nucleic  acids,  and  in  this  way,  as  shown  by  Milroy,  combina- 
tions of  nucleic  acid  and  proteids  may  be  prepared  which  behave  quite 
similar  to  true  nucleins.  All  nucleins  yield  so-called  nuclein  bases  on 
boiling  with  dilute  acids.  The  nucleins  contain  iron  to  a  considerable 
extent.     They  act  like  rather  strong  acids. 

The  nucleins  are  colorless,  amorphous,  insoluble,  or  only  slightly  soluble 
in  water.  They  are  insoluble  in  alcohol  and  ether.  They  are  more  or  less 
readily  dissolved  by  dilute  alkalies.  The  nucleins  give  the  biuret  test  and 
Millon's  reaction.  They  show  a  great  affinity  for  many  dyes,  especially 
the  basic  ones,  and  take  these  up  with  avidity  from  watery  or  alcoholic  solu- 
tions. On  burning  they  yield  an  acid  residue  which  is  very  difficult  to 
incinerate  and  which  contains  metaphosphoric  acid.  On  fusion  with  salt- 
peter and  soda  the  nucleins  yield  alkali  phosphates. 

To  prepare  nucleins  from  cells  or  tissues,  first  remove  the  chief  mass  of 
proteids  by  artificial  digestion  with  pepsin-hydrocholric  acid,  lixiviate  the 

1  Hoppe-Seyler,  Med.  chem.  Untersuch.,  4.32. 

2  Umber,  Zeitschr.  f.  klin.  Med.,  43;  Milroy,  Zeitschr.  £.  physiol.  Chem.,  22. 
8  Pfliiger's  Arch.,  47. 


126  THE  ANIMAL  CELL. 

residue  with  very  dilute  ammonia,  filter,  and  precipitate  with  hydrochloric 
acid.  The  precipitate  is  further  digested  with  gastric  juice,  washed  and 
purified  by  alternately  dissolving  in  very  faintly  alkaline  water  and 
reprecipitating  with  an  acid,  washing  with  water,  and  treating  with  alcohol- 
ether.  A  nuclein  may  be  prepared  more  simply  by  the  digestion  of  a 
nuclcoproteid.  In  the  detection  of  nucleins  we  make  use  of  the  above- 
described  method  and  testing  for  phosphorus  in  the  product  after  fusing 
with  saltpeter  and  soda.  Naturally  the  phosphates,  lecithins  (and  jecorin) 
must  first  be  removed  by  treatment  with  acid,  alcohol,  and  ether,  respec- 
tively. We  must  specially  call  attention  to  the  fact,  as  shown  by  Lieber- 
mann,1  of  the  very  great  difficulty  in  removing  lecithin  by  means  of  alcohol- 
ether.  No  exact  methods  are  known  for  the  quantitative  estimation  of 
nucleins  in  organs  or  tissues. 

Pseudonucleins  or  Paranucleins.  These  bodies  are  obtained  as  an 
insoluble  residue  on  the  digestion  of  certain  nucleoalbumins  or  phosphoglu- 
coproteids  with  pepsin-hydrochloric  acid.  Attention  is  called  to  the  fact 
that  the  pseudonuclein  may  be  dissolved  by  the  presence  of  too  much  acid 
or  by  a  too  energetic  peptic  digestion.  If  the  relationship  between  the 
degree  of  acidity  and  the  quantity  of  substance  is  not  properly  selected, 
the  formation  of  pseudonucleins  may  be  entirely  overlooked  in  the  digestion 
of  certain  nucleoalbumins.  Pseudonucleins  contain  phosphorus,  which, 
as  shown  by  Liebermann,2  is  split  off  as  metaphosphoric  acid  by  mineral 
acids. 

The  pseudonucleins  are  amorphous  bodies  insoluble  in  water,  alcohol, 
and  ether,  but  readily  soluble  in  dilute  alkalies.  They  are  not  soluble  in 
very  dilute  acids,  and  may  be  precipitated  from  their  solution  in  dilute 
alkalies  by  adding  acid.  They  give  the  proteid  reactions  very  strongly,  but 
do  not  yield  nuclein  bases. 

In  preparing  a  pseudonuclein,  dissolve  the  mother-substance  in  hydro- 
chloric acid  of  1-2  p.  m.,  filter  if  necessary,  add  pepsin  solution,  and  allow 
the  mixture  to  stand  at  the  temperature  of  the  body  for  about  twenty- 
four  hours.  The  precipitate  is  filtered  off,  washed  with  water,  and  purified 
by  alternately  dissolving  in  very  faintly  alkaline  water  and  reprecipitating 
with  acid. 

Plastin.  After  the  extraction  of  the  nucleins  from  cell  nuclei  of  certain  plants 
in  dilute  soda  solution,  a  residue  is  obtained  which  is  characterized  by  its  great 
insolubility.  The  substance  which  forms  this  residue  has  been  called  plastin. 
This  substance,  of  which  the  spongioplasm  of  the  body  of  the  cell  and  the  nucleus- 
granules  are  alleged  to  be  composed,  is  considered  as  a  nuclein  modification  of 
great  insolubility,  although  its  nature  is  not  known. 

Nucleic  Acids.  All  nucleic  acids  are  rich  in  phosphorus  and  yield  phos- 
phoric acid  and  nuclein  bases  as  cleavage  products.  The  various  nucleic 
acids  are  nevertheless  very  different  in  regard  to  the  products  they  yield. 

1  Pfliiger's  Arch.,  47. 

2  Ber.  d.  d.  chem.  Gesellsch.,  21,  and  Centralbl.  f.  d.  med.  Wissensch.,  1889. 


NUCLEIC  ACIDS. 


127 


The  nucleic  acid  from  ox-sperm  yields,  according  to  Kossel,  chiefly 
xanthine,  the  guanylic  acid  from  the  pancreas,  according  to  J'. .wo,  chiefly 
guanine,  fche  thymonucleic  acids  and  the  vegetable  nucleic  acids,  on 
the  contrary,  guanine  and  adenine  (Kossel,  Neumann,  Schmtedeberg, 

Osborne,  and  others).  In  the  vegetable  nucleic  acids,  as  far  as  we  know, 
the  pyrimidine  group  is  only  represented  by  cvtosin  and  uracil  (KOSSEL, 
Ascoli,  KOBSEL  and  Steuuel,  ( )snoRNE  and  Harris)  in  the  thymonucleic 
acids  by  cytosin,  thymin,  and  uracil  (Kossel,  Neuman,  Levene).1 
Guanylic  acid  contains  neither  uracil,  thymin,  nor  cytosin. 

The  nucleic  acids  show  a  different  composition  also  in  other  regards. 
A  pentose  group  can  be  split  off  from  guanylic  acid  and  the  vegetable 
nucleic  acids  (the  tritico-  and  yeast  nucleic  acid),  while  from  the  yeast 
nucleic  acid  also  a  hexose  is  claimed  to  be  obtained.  No  carbohydrate 
has,  on  the  contrary,  been  split  off  from  the  thymonucleic  acids.  Only 
on  deep  cleavage  have  Kossel  and  Neumann  been  able  to  obtain  levulinic 
acid  from  the  nucleic  acid  of  the  thymus  glands,  showing  that  they  contain 
a  carbohydrate  group. 

We  generally  admit  of  4  atoms  of  phosphorus  in  the  empirical  formulae 
of  the  various  nucleic  acids.  In  thymonucleic  acid  the  relationship  of 
phosphorus  to  nitrogen  is  as  4  to  14,  in  triticonucleic  acid,  4  to  16,  and  in 
guanylic  acid,  4  to  20.  The  form  of  combination  of  the  phosphorus  is  not 
known  with  positiveness,  but  it  seems  at  least  that  guanylic  and  tritico- 
nucleic acids  are  derivatives  of  a  pentahydroxylphosphoric  acid,  P(OH)5. 

All  nucleic  acids  are  amorphous,  white,  and  have  an  acid  reaction. 
They  are  readily  soluble  in  ammoniacal  or  alkaline  water  and  form  insoluble 
salts  with  the  heavy  metals,  and  as  a  rule  also  insoluble  basic  salts  with  the 
alkaline  earths.  Guanylic  acid  is  soluble  with  difficulty  in  cold  water  but 
rather  readily  in  boiling  water,  from  which  it  separates  on  cooling.  Guanylic 
acid  is  readily  precipitated  from  its  alkali  combination  by  an  excess  of 
acetic  acid.  The  other  nucleic  acids  are,  on  the  contrary,  not  precipitated 
from  such  combinations  by  an  excess  of  acetic  acid,  but  by  a  slight  excess 
of  hydrochloric  acid,  especially  in  the  presence  of  alcohol.  In  acid  solu- 
tions these  latter  nucleic  acids  give  precipitates  with  proteids,  which  are 
considered  as  nucleins.  The  behavior  of  guanylic  acid  in  this  regard  has 
not  been  shown  on  account  of  the  great  difficulty  in  dissolving  this  acid  in 

1  The  works  of  Kossel  and  his  pupils  on  nucleic  acids  are  found  in  Du  Bois-Rey- 
mond's  Arch.,  1892,  1893,  and  1894;  Sitzungsber.  d.  Berl.  Akad.  d.  Wissensch..  IS, 
1894;  Centralbl.  f.  d.  med.  Wissensch.,  1893;  Ber.  d.  deutsch.  chera.  Geeellsch.,  26 
and  27;  Zeitschr.  f.  physiol.  Chem.,  22  and  3S;  see  also  Neumann,  Arch.  f.  (Anat.  u.) 
Physiol.,  1898  and  1899,Supplb. ;  Miescher,  Hoppe-Seyler's  Med.  chem.  I'ntersuch.,  441, 
and  Arch.  f.  expt.  Path.  u.  Pharm.,  37;  Schmiedeberg,  ibid.,  37  and  43;  Osborne  and 
Harris,  Zeitschr.  f.  physiol.  Chem.,  30;  Bang,  ibid.,  26  and  31,  and  Biochem.  Centralbl., 
T,  295;  Altmann,  Arch.  f.  (Anat.  u.)  Physiol.,  1899;  Ascoli,  Zeitschr.  f.  physiol.  Chem., 
28  and  31;  Levene,  ibid.,  32,  38,  and  39. 


128  THE  ANIMAL  CELL. 

dilute  acids.  All  nucleic  acids  are  insoluble  in  alcohol  and  ether.  They 
do  not  give  either  the  biuret  test  or  Millon  's  reaction. 

Thymonucleic  Acids.1  To  this  group  belong  two  closely  related  acids 
found  in  the  thymus  gland  (Neumann).  The  nucleic  acid  in  the  salmon 
sperm  (salmonucleic  acid)  seems  to  be  identical  with  one  of  the  nucleic 
acids  of  the  thymus  gland  (Schmiedeberg,  Herlant).  The  acids  pre- 
pared by  Levene  from  the  pancreas,  spleen,  and  spermatozoa  of  the  codfish 
seem  to  be  identical,  or  at  least  closely  related  to  these.  The  nucleic  acids 
of  the  sperm  of  the  sturgeon  (Noll),  herring  (Mathews,  Gulewitsch),  and 
sea-urchin  (Mathews)2  also  probably  belong  to  this  group. 

The  salmonucleic  acid  and  the  thymusnucleic  acid  as  obtained  by 
Schmiedeberg  's  method  have  the  same  composition,  C40H56N14O16.2P2O5. 
On  heating  the  free  acid  with  water  at  the  water-bath  temperature  there  has 
been  split  off  besides  adenine  and  guanine  a  new  acid,  thymic  acid,  which 
is  readily  soluble  in  water  and  which  yields  a  barium  salt  which  is  also 
soluble  in  water,  C16H23N3P2012Ba  (Kossel  and  Neumann).  On  hydro- 
lytic  cleavage  with  sulphuric  acid  Kossel  and  Neumann  obtained  phos- 
phoric acid  (about  23  per  cent  P205),  thymin  (8  per  cent),  levulinic  acid, 
cytosin,  ammonia,  guanine,  and  adenine  from  thymusnucleic  acid. 

Guanylic  Acid.  This  acid,  which  thus  far  has  only  been  obtained  from 
the  pancreas,  has,  according  to  Bang,  the  composition  C44H66N20P4O34.  It 
is  readily  soluble  in  warm  water,  but  partially  separates  out  on  cooling. 
It  is  considered  as  an?  ester  of  a  glycerophosphoric  acid  and  decomposes 
on  hydrolytic  cleavage  with  acids,  according  to  Bang,  into  4  molecules 
of  guanine,  3  molecules  of  pentose  (1-xylose  according  to  Neuberg),  3 
molecules  of  glycerine,  and  4  molecules  of  phosphoric  acid. 

According  to  the  more  recent  investigations  of  Bang  and  Kaaschou  3 
the  guanylic  acid  which  Bang  now  designates  as  /?-acid  is  formed,  in  the 
preparation  from  another  acid  called  a-guanylic  acid,  by  the  action  of 
the  alkali.  The  a-guanylic  acid,  which  is  readily  soluble  in  water,  even 
in  cold  water,  contains  less  phosphorus  and  nitrogen  (6.65  and  15.38  per 
cent  respectively)  as  compared  to  the  /3-acid,  which  contains  7.64  per  cent 
phosphorus  and  18.21  per  cent  nitrogen.  By  the  action  of  alkalies  the 
a-guanylic  acid  splits  off  a  pentose  group  and  is  converted  into  the  /?-acid. 

The  following  acid  is  also  generally  included  among  the  nucleic  acids : 

Inosinic  acid,  Ci0H13N4POg,  was  first  isolated  by  Liebig  from  the  flesh  of 
certain  animals  and  then  closely  studied  by  Haiser.4  It  contains  phosphorus, 
is   amorphous,  and  gives  crystalline  salts  with  barium   and   calcium.      Haiser 

1  This  name  will  be  used  as  a  group  name  for  all  nucleic  acids  closely  related  to 
the  thymusnucleic  acids. 

2Herlant,  Arch.  f.  expt.  Path.  u.  Pharm.,  44;  Noll,  Zeitschr.  f.  physiol.  Chem.,  25; 
Mathews,  ibid.,  23;  Gulewitsch,  ibid.,  27. 

8  Hofmeister's  Beitrage,  4. 

4  Liebig,  Annal.  d.  Chem.  u.  Pharm.,  62j  F.  Haiser,  Monatshefte  f.  Chem.,  16. 


NUCLEIC  ACIDS.  1-0 

obtained  hypoxanthine  as  a  cleavage  product  and  probably  also  trioxyvalerianic 
acid,  though  it  has  not  been  positively  proven. 

The  thymusnucleic  acida  may  be  prepared  as  the  copper  salt,  according 
to  ScHMlEDEBERG,  from  the  heads  of  the  salmon  spermatozoa  or  from  the 
residue  after  the  peptic  digestion  of  the  thymus  glands  (Herlant).  The 
protamins  are  removed  by  the  action  of  copper  chloride  and  the  lasl  traces 
of  proteid  removed  by  dissolving  the  residue  in  dilute  caustic  potash  and 
precipitating  this  solution  with  alcohol,  and  this  is  repeated  until  it  fails  to 
give  the  biuret  test.  The  copper  salt  can  be  precipitated  by  copper  chloride 
from  the  watery  solution  of  the  potassium  nucleate  after  acidification  with 
acetic  acid.  According  to  Neumann  the  two  thymusnucleic  acids,  a  and  /?, 
can  be  obtained  from  the  gland,  after  previously  boiling  the  same  with 
water  containing  acetic  acid  and  then  cutting  it  up  fine.  The  finely 
divided  gland  is  boiled  with  alkaline  water  (about  3  per  cent  NaOH)  for 
one-half  hour  for  acid  a  and  two  hours  for  acid  t3,  and  sodium  acetate  added 
at  the  same  time.  After  neutralization  with  acetic  acid,  filter,  concentrate, 
and  precipitate  with  alcohol.  The  nucleic  acids  can  be  obtained  from 
the  precipitated  sodium  salts  of  the  nucleic  acid  by  precipitating  with 
alcohol  containing  hydrochloric  acid.  Levene's  *  method  consists,  on 
the  contrary,  in  treating  the  organs  first  with  5  per  cent  sodium  hydrate 
or  with  S  per  cent  ammonia  in  the  cold,  then  nearly  neutralizing  with 
acetic  acid,  precipitating  the  proteids  with  picric  acid,  and  treating  the 
strongly  acidified  liquid  (acetic  acid)  with  alcohol.  In  the  presence  of 
sufficient  acetate  the  nucleic  acids  are  precipitated. 

Guanylic  acid  may  be  best  prepared  according  to  Bang  and  Raaschou 
by  the  following  method:  After  treating  the  pancreas  with  1  per  cent 
sodium  hydrate  solution  for  twenty-four  hours  at  the  room  temperature 
it  is  dissolved  by  warming,  then  neutralized  by  acetic  acid  anil  made  faintly 
acid,  filtered,  made  faintly  alkaline  with  ammonia,  strongly  concentrated, 
and  precipitated  with  alcohol  while  hot.  The  proteoses  remain  in  solu- 
tion and  the  precipitated  guanylic  acid  (a-acid)  is  purified  by  repeated 
solution  in  water  and  precipitation  by  alcohol. 

Plant  Nucleic  Acids.  Those  best  known  are  the  yeast  nucleic  acid  and  the  tri- 
ticonucleic  acid,  C^H^X,,?^,,  isolated  by  Osbokxe  and  Harris  from  the  wheat 
embryo,  and  which  according  to  these  investigators  is  identical  with  the  yeast 
nucleic  acid.  The  plant  nucleic  acids  are  nearly  related  to  the  thymonueleic  acids, 
but  differ  from  them  not  only  by  the  presence  of  the  pentose  groups,  but  also  by 
the  fact  that  in  the  thymonueleic  acids  the  pyrimidine  groups  are  represented  by 
uracil,  eytosin,  and  thymin,  and  in  the  triticonucleic  acid  by  cytosin  and  uracil. 

This  last  acid,  which  is  dextrorotatory,  yields  on  hydrolysis  with  add  1  molecule 
of  guanine,  1  molecule  of  adenine  and  cytosin  (Wheeler  and  JOHNSON*),  2  mole- 
cules of  uracil,  and  3  molecules  of  pentose  for  every  4  atoms  of  phosphorus. 
Levexe  has  been  able  to  prepare  from  the  tubercle  bacilli  nucleic  acids  whose 
nature  has  not  been  closely  studied. 

Plasminic   acid  is  an  acid  which  was  prepared  by  Ascoir  and  Kossels  by 

1  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  43;  Herlant,  ibid.,  44;  Neumann, 
Arch.  f.  (Anat.  u.)  Physiol.,  1S99,  Supplbl.;  Levene,  Zeitschr.  f.  physiol.  Chem.,  32; 
Ixostytsehew,  ibid.,  39. 

3  Amer.  Chem.  Journ.,  29. 

s  Ascoli,  Zeitschr.  I.  physiol.  Chem.,  2S. 


130  THE  ANIMAL  CELL. 

the  action  of  alkali  upon  yeast.  It  contains  iron  and  is  soluble  in  very  dilute 
hydrochloric  acid  (1  p.  m.).  It  is  still  a  question  whether  it  is  a  mixture  or  a 
chemical  individual. 

In  regard  to  the  preparation  of  yeast  and  triticonucleic  acid  we  must  refer  to 
the  works  of  Altmann,  Kossel,  Osborne  and  Harris.1 

Among  the  cleavage  products  of  the  nucleic  acids  the  purin  deriva- 
tives and  the  pyrimidine  derivatives  are  of  special  interest. 

Purin  Bases  (nuclein  bases,  alloxuric  bases,  xanthine  bodies).  With, 
these  names  we  designate  a  group  of  bodies  consisting  of  carbon,  hydrogen, 
nitrogen,  and  in  most  cases  also  of  oxygen,  which,  by  their  composition,, 
show  a  relationship  not  only  among  themselves,  but  also  with  uric  acid. 
All  these  bodies,  uric  acid  included,  are  considered  as  consisting  of  an. 
alloxuric  and  a  urea  nucleus,  and  for  this  reason  Kossel  and  Krtjger  have 
called  them  alloxuric  bases,  or  the  entire  group,  including  uric  acid,  alloxuric 
bodies.  According  to  E.  Fischer,2  who  has  not  only  shown,  in  several 
ways,  the  close  relationship  of  uric  acid  to  this  group,  but  has  also  prepared 
a  number  of  the  members  of  this  group  synthetically,  they  are  all  derived 

N=CH 

HC     C— NH 

from  a  compound,  C5H4N4=      II      II  >CH,  called  purin. 

N— C—  N  ^ 

The  different  purin  bodies  are  derived  therefrom  by  the  substitution  of  the 
various  hydrogen  atoms  by  hydro xyl,  amid,  or  alkyl  groups.  In  order  to  signify 
the  different  positions  of  substitution  Fischer  has  proposed  to  number  the  nine 
members  of  the  purin  nucleus  in  the  following  way: 

IN— C6 

I       I 
2C   50—  N7 

I       I        >C8. 
3N— 0— N9 
4 

HN— CO 

CO  C—  NH 

For  example,  uric  acid,       I      jl       TTJ  >  CO,  is   2,  6,    8-trioxypurin,  adenine 
HN — C — NH 
N-C.NH2  HN— CO 

II  II 

HC     C — N  CO  C— N  .CH, 

i'r     J!     ,.  '  >CH  =  6-aminopurin,      and     heteroxanthine  __J_     Ji     ,T>~„  = 
N — C — NH  r  HN— C— N    CH 

7-methyl-  2,  6-dioxypurin,  etc. 

The  starting-point  used  by  Fischer  for  the  synthetical  preparation  of  the 
purin  bases  was  2,  6,  8-trichlorpurin,  which  is  obtained,  with  8-oxy-2,  6-dichlor- 
purin  as  intermediary  products,  from  potassium  urate  and  phosphorus  oxychlor- 
ide.     The  close  relation  between  uric  acid  and  the  nuclein  bases  follows  from 


1  See  foot-note  1 ,  page  1 27. 

'See  Fischer,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30  and  32. 


I'r LIS   BASES.  131 

the  fact,  as  shown  by  Srxnvuc,1  that  two  bodies  may  be  obtained  on  t ho  reduction 
of  uric  acid  in  alkaline  solution,  which,  although  not  quite  identical  with  xanthine 
and  hypoxanthine,  are  at  leaal  very  similar  thereto.  GaTJTOBB1  claims  to  have 
prepared    xanthine  synthetically   by   heating   hydrocyanic   acid   with   water   and 

acetic  acid. 

The  purin  bodies  or  alloxuric  bodies  found  in  t lie  animal  body  or  its 
excreta  are  as  follows:  Uric  acid,  xanthiru  ,  In  U  roxanthiru  ,  l-methylxanihiiu 
paraxanthine,  guanine,  epiguanine,  hypoxanihine,  episarkine,  adenine,  and 
ne.     The  bodies  theobromine,  theophylline,  and  caffeine  occurring  in  the 

vegetable  kingdom  stand  in  close  relationship  to  this  group. 

The  composition  of  the  most  important  purin  bodies  from  a  physio- 
logical standpoint  is  as  follows: 

Uric  acid,  C5H4X403 2,6,  8-trioxypurin 

Xanthine,  CjH«N40, 2,  G-dioxypurin 

1  -rmthylxanthine,  CJELN40, 1-methyl 

roxanthine,  CeHsX40, 7-      " 

Theophylline,  <-H,\,<  >2 1,3-dimethyl 

Paraxanthine,  </-llsX4<  >, 1,7- 

Theobroraine,  r.H.X4o, 3,7- 

ine,  C\H,„X4C)2 1,3,  7-trimethvl 

Hypoxanthine,  CsH4X40 6-oxypurin 

( hianine,  C,H,X,0 2-amino       " 

Epiguanine,  CI0H13X9O2 7-methyl  "      "  "        " 

Adenine,  C5H5X5 6-amii.^nirin 

Episarkine,  C4H,X403(?) 

Carnine,  C7HsX403 

After  Salomon  3  had  shown  the  occurrence  of  xanthine  bodies  in  young 
cells  the  importance  of  the  xanthine  bodies  as  decomposition  products  of  cell 
nuclei  and  of  nucleins  was  shown  by  the  pioneering  researches  of  Kossel, 
who  discovered  adenine  and  theophylline.  Kossel  gave  them  the  name 
i;:elein  bases.  In  those  tissues  in  which,  as  in  the  glands,  the  cells  have 
kept  their  original  state  the  nuclein  bases  are  not  found  free,  but  in  com- 
bination with  other  atomic  groups  (nucleins).  In  such  tissue,  on  the 
contrary,  as  in  muscles,  which  are  poor  in  cell  nuclei,  the  nuclein  bases  are 
found  in  the  free  state.  Since  the  nuclein  bases,  as  suggested  by  Kossel, 
stand  in  close  relationship  to  the  cell  nucleus,  it  is  easy  to  understand  why 
the  quantity  of  these  bodies  is  so  greatly  increased  when  large  quantities  of 
nucleated  cells  appear  in  such  places  as  were  before  relatively  poorly 
endowed.  As  an  example  of  this,  the  blood,  in  leucaemia,  is  extremely 
rich  in  leucocytes.  In  such  blood  Kossel4  found  1.04  p.  m.  nuclein  bases, 
against  only  traces  in  the  normal  blood.     That  the  nuclein  bases  are  also 

1  Zeitschr.  f.  physiol.  Chem.,  23. 

1  Compt.  rend.,  9S,  1523,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  31.  In  regard  to 
the  synthesis  of  uric  acid  and  purin  bases  see  Traube,  ibid.,  33. 

3  Sitzunsber.  d.  Bot.  Verein  der  Provinz  Brandenburg,  1SS0 

4  Zeitschr.  f.  physiol.  Chom.,  7. 


132  THE  ANIMAL  CELL. 

intermediate  steps  in  the  formation  of  urea  or  uric  acid  in  the  animal 
organism  is  probable,  and  will  be  shown  later  (see  Chapter  XV). 

Only  a  few  of  the  nuclein  bases  have  been  found  in  the  urine  or  in  the 
muscles.  Only  four  bases — xanthine,  guanine,  hypoxanthine,  and  adenine — 
have  been  obtained,  thus  far,  as  cleavage  products  of  nucleins.  In  regard 
to  the  other  purin  bodies  we  refer  the  reader  to  their  respective  chapters. 
Only  the  above  four  bodies,  the  real  nuclein  bases,  will  be  considered  at  this 
time. 

Of  these  four  bodies  xanthine  and  guanine  form  one  special  group  and 
hypoxanthine  and  adenine  another.  By  the  action  of  nitrous  acid  guanine  is 
converted  into  xanthine  and  adenine  into  hypoxanthine. 

C5H4N4O.NH+  HN02  =  C5H4N402+  N2+  H20 ; 

Guanine  Xanthine 

C5H4N4.NH+  HN02  =  C5H4N40  +  N2+  H20. 

Adenine  Hypoxanthine 

By  putrefaction  guanine  is  converted  into  xanthine  and  adenine  into  hypo- 
xanthine. On  cleavage  with  hydrochloric  acid  all  four  of  the  bodies  are  con- 
verted into  ammonia,  glycocoll,  carbon  dioxide,  and  formic  acid.  On  oxidation 
with  hydrochloric  acid  and  potassium  chlorate,  xanthine,  bromadenine,  and  brom- 
hypoxanthine  yield  alloxan  and  urea;  guanine  yields  guanidine,  parabanic  acid 
(an  oxidation  product  of  alloxan),  and  carbon  dioxide. 

The  nuclein  bases  form  crystalline  salts  with  mineral  acids,  which  are 
decomposed  by  water  with  the  exception  of  the  adenine  salts.  They  are 
easily  dissolved  by  alkalies,  while  with  ammonia  their  action  is  somewhat 
different.  They  are  all  precipitated  from  acid  solution  by  phosphotungstic 
acid;  they  also  separate  as  a  silver  combination  on  the  addition  of  ammonia 
and  ammoniacal  silver-nitrate  solution.  These  precipitates  are  soluble  in 
boiling  nitric  acid  of  1.1  specific  gravity.  All  xanthine  bodies  are  also  pre- 
cipitated by  Fehling's  solution  (see  Chapter  XV)  in  the  presence  of  a 
reducing  substance  such  as  hydroxylamine  (Drechsel  and  Balke).  Copper 
sulphate  and  sodium  bisulphite  may  also  be  used  to  advantage  in  their 
precipitation  (Kruger1).  This  behavior  of  the  xanthine  bases  is  made- 
use  of  to  the  same  advantage  as  the  silver  solution  in  their  precipitation, 
and  preparation. 

HN— CO 

CO  C— NH 
Xanthine,  C5H4N402=H L _n_  N~>CH  (2>  6-dioxypurin),  is  found  in: 

the  muscles,  liver,  spleen,  pancreas,  kidneys,  testicles,  carp-sperm,  thymus, 
and  brain.  It  occurs  in  small  quantities  as  a  physiological  constitu- 
ent of  urine,  and  it  occasionally  has  been  found  as  a  urinary  sediment,  or 

1  Balke,  zur  Kenntniss  der  Xanthinkorper,  Inaug.-Diss.  Leipzig,  1893 ;  Kruger, 
Zeitschr.  f.  physiol.  Chem.,  18. 


XANTHINE.  133 

calculus.     It  was  first  observed  in  such  a  stone  by  Makcet.     Xanthine  is 
found  in  larger  amounts  in  a  few  varieties  of  guano  (Jarvia  guano). 

Xanthine  is  amorphous,  or  forms  granular  masses  of  crytals,  or  may  also, 
according  to  Horhaczewski,'  separate  as  masses  of  shining,  thin,  large 
rhombic  plates  with  1  mol.  water  of  crystallization.  It  is  very  slight  In- 
soluble in  water,  in  14,151-14,600  parts  at  16°  C,  and  in  1300-1500 
parts  at  100°  C.  (Almen  ■).  It  is  insoluble  in  alcohol  or  ether,  but  is 
readily  dissolved  by  alkalies  and  with  difficulty  by  dilute  acids.  With 
hydrochloric  acid  it  gives  a  crystalline,  difficultly  soluble  combination. 
With  very  little  caustic  soda  it  gives  a  readily  crystallizable  compound. 
which  is  easily  dissolved  by  an  excess  of  alkali.  Xanthine  dissolved  in 
ammonia  gives  with  silver  nitrate  an  insoluble,  gelatinous  precipitate  of 
xanthine  silver.  This  precipitate  is  dissolved  by  hot  nitric  acid,  and  by  this 
means  an  easily  soluble  crystalline  double  combination  is  formed.  A 
watery  xanthine  solution  is  precipitated  on  boiling  with  copper  acetate.  At 
ordinary  temperatures  xanthine  is  precipitated  by  mercuric  chloride  and  by 
ammoniacal  basic  lead  acetate.  It  is  not  precipitated  with  basic  lead 
acetate  alone. 

When  evaporated  to  dryness  in  a  porcelain  dish  with  nitric  acid,  xanthine 
§  ives  a  yellow  residue,  which  turns,  on  the  addition  of  caustic  soda,  first 
red.  and,  after  heating,  purple-red.  If  we  place  some  chloride  of  lime  with 
some  caustic  soda  in  a  porcelain  dish  and  add  the  xanthine  to  this  mixture, 
at  first  a  dark-green  and  then  quickly  a  brownish  halo  forms  around  the 
xanthine  grains  and  finally  disappears  (Hoppe-Seyler).  If  xanthine  be 
warmed  in  a  small  vessel  on  the  water-bath  with  chlorine-water  and  a  trace 
of  nitric  acid  and  evaporated  to  dryness,  and  the  residue  is  then  exposed  under 
a  bell- jar  to  the  vapors  of  ammonia,  a  red  or  purple-violet  color  is  produced 
(Weidel's  reaction).  E.  Fischer  3  has  modified  Weidel's  reaction  in  the 
following  way.  He  boils  the  xanthine  in  a  test-tube  with  chlorine-water  or 
with  hydrochloric  acid  and  a  little  potassium  chlorate,  then  evaporates  the 
liquid  carefully  and  moistens  the  dry  residue  with  ammonia. 

HN— CO 

I       I 
Guanine,    C5H5N50  =  H2X.C     C— NH 

\r     r    m*^  ^H     &  "  am*no  "  6"  oxypurin). 

IN       vv      i\ 

Guanine  is  found  in  organs  rich  in  cells,  such  as  the  liver,  spleen,  pancreas, 
testicles,  and  in  salmon-sperm.  It  is  further  found  in  the  muscles  (in  very 
small  amounts),  in  the  scales  and  in  the  air-bladder  of  certain  fishes  as 
iridescent  crystals  of  guanine-lime;  in  the  retinal  epithelium  of  fishes,  in 
guano,  and  in  the  excrement  of  spiders  it  is  found  as  chief  constituent.     It 

1  Zeitschr.  f.  physiol.  Chem.,  23. 

1  Journ.  f.  prakt.  Chem.,  96. 

5  Ber.  d.  deutsch.  chem.  Gesellsch.,  30,  2236. 


134  THE  ANIMAL  CELL. 

also  occurs  in  human  and  pig  urine.  Under  pathological  conditions  it  has 
been  found  in  leucaemic  blood,  and  in  the  muscles,  ligaments,  and  articula- 
tions of  pigs  with  guanine-gout. 

Guanine  is  a  colorless,  ordinarily  amorphous  powder  which  may  be 
obtained  as  small  crystals  by  allowing  its  solution  in  concentrated  ammonia 
to  spontaneously  evaporate.  According  to  Horbaczewski  it  may  under 
certain  conditions  appear  in  crystals,  similar  to  creatinine  zinc  chloride. 
It  is  insoluble  in  water,  alcohol,  and  ether.  It  is  rather  easily  dissolved 
by  mineral  acids  and  readily  by  alkalies,  but  it  dissolves  with  great  difficulty 
in  ammonia.  According  to  Wulff  1  100  c.  c.  of  cold  ammonia  solution 
containing  1,  3,  and  5  per  cent  NH3  dissolve  9,  15,  and  19  milligrams  of 
guanine  respectively.  The  solubility  is  relatively  increased  in  hot  ammonia 
solution.  The  hydrochloride  readily  crystallizes,  and  this  has  been  recom- 
mended by  Kossel2  in  the  microscopical  detection  of  guanine  on  account 
of  its  behavior  to  polarized  light.  The  sulphate  contains  2  molecules  of  water 
of  crystallization,  which  is  completely  expelled  on  heating  to  120°  C,  and  for 
this  reason  as  well  as  the  fact  that  guanine  yields  guanidine  on  decomposition 
with  chlorine-water,  differentiates  it  from  6-amino-2-oxypurin,  which  is  con- 
sidered as  an  oxidation  product  of  adenine  and  possibly  occurs  as  a  chem- 
ical metabolic  product  (E.  Fischer).  The  6-amino-2-oxypurin  sulphate 
contains  only  1  molecule  of  water  of  crystallization,  which  is  not  expelled  at 
120°  C.  Very  dilute  guanine  solutions  are  precipitated  by  both  picric 
acid  and  metaphosphoric  acid.  These  precipitates  may  be  used  in  the 
quantitative  estimation  of  guanine.  The  silver  combination  dissolves  with 
difficulty  in  boiling  nitric  acid,  and  on  cooling  the  double  combination 
crystallizes  out  readily.  Guanine  acts  like  xanthine  in  the  nitric-acid  test, 
but  gives  with  alkalies  on  heating  a  more  bluish-violet  color.  A  warm 
solution  of  guanine  hydrochloride  gives  with  a  cold  saturated  solution  of 
picric  acid  a  yellow  precipitate  consisting  of  silky  needles  (Capranica). 
With  a  concentrated  solution  of  potassium  bichromate  a  guanine  solution 
gives  a  crystalline,  orange-red  precipitate,  and  with  a  concentrated  solu- 
tion of  potassium  ferricyanide  a  yellowish-brown,  crystalline  precipitate 
(Capranica).  The  composition  of  these  and  other  guanine  combinations 
has  been  studied  by  Kossel  and  Wulff.3    Guanine  does  not  give  Weidel  'a 

reaction. 

HN— CO 

I  I 

Hypoxanthine,    Sarkin,  C5H4N40=HC    C— NH  =  (6-oxypunn). 

II  II  >CH 

N— C— N 


1  Zeitschr.  f.  physiol.  Chem.,  17. 

2  Ueber  die  chem.  Zusammensetz  der  Zelle,  Verb.,  d.  physiol.  Gesellsch.  zu  Berlin, 
1890-91,  Nos.  5  and  6. 

»  Zeitschr.  f.  physiol.  Chem.,  17;  Capranica,  ibid.,  4. 


IIYPOXANTHINE,  ADENINE.  135 

This  body  is  found  in  the  same  tissues  as  xanthine.  It  is  especially  abund- 
ant in  the  sperm  of  the  salmon  and  carp.  Hypoxanthine  occurs  also  in 
the  marrow  and  in  very  small  quantities  in  normal  urine,  and,  as  it  seems, 
also  in  milk.  It  is  found  in  rather  considerable  quantities  in  the  blood 
and  urine  in  leucaemia. 

Hypoxanthine~forms  very  small,  colorless,  crystalline  needles.  It  dissolves 
with  difficulty  in  cold  water,  but  the  statements  in  regard  to  the  solubility 
therein  are  very  contradictor}'.1  It  dissolves  more  readily  in  boiling  water, 
in  about  70-SO  parts.  It  is  nearly  insoluble  in  alcohol,  but  is  dissolved  by 
acids  and  alkalies.  The  combination  with  hydrochloric  acid  is  crystalline, 
and  is  more  soluble  than  the  corresponding  xanthine  combination.  It 
is  easily  soluble  in  dilute  alkalies  and  ammonia.  The  silver  combina- 
tion dissolves  with  difficulty  in  boiling  nitric  acid.  On  cooling  a  mixture 
of  two  hypoxanthine  silver-nitrate  compounds  possessing  an  inconstant 
composition  separates  out.  On  treating  this  mixture  with  ammonia  and 
an  excess  of  silver-nitrate  and  heating,  a  hypoxanthine-silver  combina- 
tion is  formed  which  when  dried  at  120°  C.  has  a  constant  composition, 
2(C5H2Ag2N40)H20,  and  is  used  in  the  quantitative  estimation  of  hypo- 
xanthine. Hypoxanthine  picrate  is  soluble  with  difficulty,  but  if  a  boiling- 
hot  solution  of  the  same  is  treated  with  a  neutral  or  only  faintly  acid 
solution  of  silver  nitrate  the  hypoxanthine  is  nearly  quantitatively  precipi- 
tated as  the  compound  C5H3AgX4O.C6H2(X02)3OH.  Hypoxanthine  does  not 
yield  an  insoluble  compound  with  metaphosphoric  acid.  When  treated, 
like  xanthine,  with  nitric  acid  it  yields  a  nearly  colorless  residue  which 
on  warming  with  alkali  does  not  turn  red.  Hypoxanthine  does  not  give 
Weidel's  reaction.  After  the  action  of  hydrochloric  acid  and  zinc  a 
hypoxanthine  solution  becomes  first  ruby-red  and  then  brownish-red  in 
color  on  the  addition  of  an  excess  of  alkali  (Kossel).  According  to  E. 
Fischer  2  a  red  coloration  occurs  even  in  the  acid  solution. 
N=C.NH2 

I      I 
Adenine,  C5H5N5=HC    C— NHv 

X?H  (6-aminopurin),    was  first    found 

N— C—  N  /        y  l        h 

by  Kossel  3  in  the  pancreas.  It  occurs  in  all  nucleated  cells,  but  in 
greatest  quantities  in  the  sperm  of  the  carp  and  in  the  thymus.  Adenine 
has  also  been  found  in  leucaemic  urine  (Stadthagen  4).  It  may  be  obtained 
in  large  quantities  from  tea-leaves. 

Adenine  crystallizes  with  3  molecules  of  water  of  crystallization  in  long 
needles  which  become  opaque  gradually  in  the  air,  but  much  more  rapidly 

1  See  E.  Fischer,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30. 
'Kossel,  Zeitschr.  f.  physiol.  Chem.,  12,  252;  E.  Fischer,  1.  c. 
sSee  Zeitschr.  f.  physiol.  Chem.,  10  and  12. 
'Yirchow's  Arch.,  109. 


136  THE  ANIMAL   CELL. 

when  warmed.  If  the  crystals  are  warmed  slowly  with  a  quantity  of 
water  insufficient  for  solution,  they  become  suddenly  cloudy  at  53°  C,  a 
characteristic  reaction  for  adenine.  It  dissolves  in  1086  parts  cold  water, 
but  is  easily  soluble  in  warm.  It  is  insoluble  in  ether,  but  somewhat  soluble 
in  hot  alcohol.  Adenine  is  easily  soluble  in  acids  and  alkalies.  It  is  more 
easily  soluble  in  ammonia  solution  than  guanine,  but  less  soluble  than 
hypoxanthine.  The  silver  combination  of  adenine  is  difficultly  soluble  in 
warm  nitric  acid,  and  deposits  on  cooling  as  a  crystalline  mixture  of 
adenine  silver-nitrate.  With  picric  acid  adenine  forms  a  compound, 
C5H5N5.C6H2(N02)3OH,  which  is  very  insoluble  and  which  separates  more 
readily  than  the  hypoxanthine  picrate  and  which  can  be  used  in  the  quanti- 
tative estimation  of  adenine.  We  also  have  an  adenine  mercury-picrate. 
Adenine  gives  a  precipitate  which  dissolves  in  an  excess  of  the  acid  with 
metaphosphoric  acid  if  the  solution  is  not  too  dilute.  Adenine  hydro- 
chloride gives  with  gold  chloride  a  double  combination  which  consists 
in  part  of  leaf-shaped  aggregations  and  in  part  of  cubical  or  prismatic 
crystals,  often  with  rounded  corners.  This  compound  is  used  in  the  micro- 
scopic detection  of  adenine.  With  the  nitric-acid  test  and  with  Weidel's 
reaction  adenine  acts  in  the  same  way  as  hypoxanthine.  The  same  is  true 
for  its  behavior  to  hydrochloric  acid  and  zinc  and  subsequent  addition 
of  alkali. 

The  principle  for  the  preparation  and  detection  of  the  four  above- 
described  xanthine  bodies  in  organs  and  tissues  is,  according  to  Kossel  and 
his  pupils,  as  follows:  The  finely  divided  organ  or  tissue  is  boiled  for  three 
or  four  hours  with  sulphuric  acid  of  about  5  p.  m.  The  filtered  liquid  is 
freed  from  proteid  by  basic  lead  acetate,  and  the  new  filtrate  is  treated  with 
sulphuretted  hydrogen  to  remove  the  lead,  again  filtered,  concentrated, 
and,  after  adding  an  excess  of  ammonia,  precipitated  with  ammoniacal 
silver  nitrate.  The  silver  combination  (with  the  addition  of  some  urea  to 
prevent  nitrification)  is  dissolved  in  not  too  large  a  quantity  of  boiling  nitric 
acid  of  sp.  gr.  1.1,  and  this  solution  filtered  boiling  hot.  On  cooling  the 
xanthine-silver  remains  in  the  solution,  while  the  double  combination  of 
guanine,  hypoxanthine,  and  adenine  crystallizes  out.  The  xanthine-silver 
may  be  precipitated  from  the  filtrate  by  the  addition  of  ammonia  and  the 
xanthine  set  free  by  means  of  sulphuretted  hydrogen.  The  three  above- 
mentioned  silver-nitrate  combinations  are  decomposed  in  water  with 
ammonium  sulphide  and  heat;  the  silver  sulphide  is  filtered  off,  the  filtrate 
concentrated,  saturated  with  ammonia,  and  digested  on  the  water-bath. 
The  guanine  remains  undissolved,  while  the  other  two  bases  pass  into  solu- 
tion. A  part  of  the  guanine  is  still  retained  by  the  silver  sulphide,  and  may 
be  liberated  by  boiling  it  with  dilute  hydrochloric  acid  and  then  saturating 
the  filtrate  with  ammonia.  When  the  above  filtrate  containing  the  adenine 
and  hypoxanthine,  which  has  been,  if  necessary,  freed  from  ammonia  by 
evaporation,  is  allowed  to  cool,  the  adenine  separates,  while  the  hypoxanthine 
remains  in  solution.    According  to  Balke  1  we  can  to  advantage  precipitate 

'L.c. 


ESTIMATION  OF   THE  NUCLEIN  BASES.  137 

the  xanthine  bases  with  copper  salt  and  hydroxylamine  as  above  mentioned 
and  then  further  separate  the  bodies. 

The  method  of  Buhiax  and  Hall1  Is  serviceable  in  the  estimation  of  the 
tot;il  quantity  of  purin  bodies  in  animal  organs;  the  quantitative  estima- 
tion of  the  various  bases  is  performed  in  the  main  according  to  the  method 
above  described.  ..The  xanthine  is  weighed  as  xanthine  silver.  The  three 
silver-nitrate  compounds  are  converted  into  the  corresponding  silver  com- 
binations by  ammonia  and  the  addition  of  silver  nitrate  and  then  ammonium 
sulphide  allowed  to  act  upon  the  carefully  washed  silver  compounds.  The 
guanine  is  weighed  as  such.  The  ammoniacal  filtrate  containing  the  adenine 
and  hypoxanthine,  which  must  not  be  mixed  with  the  hydrochloric-acid 
extract  of  the  silver  sulphide,  is  neutralized  and  a  cold  concentrated  solu- 
tion of  sodium  picrate  added  until  the  entire  liquid  has  a  pronouncedly 
yellow  color.  The  adenine  picrate  is  immediately  filtered  off,  washed  on 
the  filter  paper  with  water,  dried  at  above  100°  C,  and  weighed.  The  fil- 
trate containing  the  hypoxanthine  is  gradually  treated  while  boiling  hot 
with  silver  nitrate  and  after  cooling  more  silver  nitrate  is  added  to  see  if 
the  precipitation  is  complete.  The  hypoxanthine-silver  picrate  is  washed, 
dried  at  100°  C,  and  weighed.  In  regard  to  the  composition  of  these 
compounds  see  pages  134  and  145  This  method  of  separating  adenine 
and  hypoxanthine  presupposes  the  absence  of  hydrochloric  acid  in  the 
liquid. 

The  above  method  of  separation  with  ammonia  does  not  give  exact 
results  on  account  of  the  not  inconsiderable  solubility  of  guanine  in  warm 
ammonia.  According  to  Kossel  and  Wulff  2  the  guanine  may  therefore 
be  precipitated  from  sufficiently  dilute  solutions  by  an  excess  of  metaphos- 
phoric  acid  and  the  nitrogen  determined  in  the  washed  precipitate  by 
K.ikldahl's  method.  The  adenine  and  hypoxanthine  may  be  precipitated 
from  the  filtrate  by  ammoniacal  silver  nitrate.  The  silver  compound  is 
decomposed  with  very  dilute  hydiochloric  acid  and  the  adenine  separated 
from  the  hypoxanthine  according  to  the  suggestion  of  Bruhns.3  In  regard 
to  the  complications  in  the  detection  and  exact  estimation  of  purin  bodies 
in  organ  extracts  we  refer  to  the  works  of  His  and  Hagen  and  Burian  and 
Hall.4 

NH— €0 

Uracil,  C4H4N202  =  CO      CH  (2, 6-dioxypyrimidine) ,  was  first  obtained  by 

NH— CH 

Ascoli  and  Kossel  from  yeast  nucleic  acid  and  later  prepared  by  Kossel 
and  Steudel  from  thymusnucleic  acid  and  herring  testicles  and  by 
Levene  from  the  spleen  and  pancreas  nucleic  acids.  The  synthetical 
preparation  was  first  performed  by  E.  Fischer  and  Roeder.5 


1  Zeitschr.  f.  physiol.  Chem.,  3S. 
3  Ibid.,  17. 

*  Ibid.,  U,  559. 

*  His  and  Hagen,  ibid.,  30,  and  Burian  and  Hall,  ibid.,  38. 

6  Ascoli,  ibid.,  31;    Kossel  and  Steudel,  ibid.,  37;    Levene,  ibid.,  38,  39;  E.  Fischer 
and  Roeder,  Ber.  d.  d.  chem.  Gesellsch.,  34. 


138  THE  ANIMAL  CELL. 

Uracil  crystallizes  in  rosette-formed  needles.  On  careful  heating  it 
sublimes  partly  undecomposed,  but  develops  red  vapors  and  decomposes 
in  part.  It  is  readily  soluble  in  hot  water  and  less  so  in  cold  water  and 
is  nearly  insoluble  in  alcohol  and  ether.  It  is  readily  soluble  in  ammonia. 
It  is  only  precipitated  by  silver-nitrate  solution  after  the  careful  addition 
of  ammonia  or  baryta-water,  as  the  precipitate  is  readily  soluble  in  an 
excess  of  ammonia.  Uracil  responds  to  Weidel's  test  (p.  133).  In  re- 
gard to  the  preparation  of  uracil  see  Kossel  and  Steudel.1 

NH— CO 

I  I 

Thymin,  C5H6N20,  =  CO      C.CH3  (5-methyluracil).    This  body,  which  is 

I  II 

NH— CH 

identical  with  nucleosin  obtained  by  Schmiedeberg  from  salmonucleic  acid, 

is  obtained  from  the  thymusnucleic  acids  and  was  first  prepared  by  Kossel 

and  Neumann  from  thymusnucleic  acid.      E.  Fischer  and  Roeder  2  have 

prepared  it  synthetically. 

Thymin  crystallizes  in  stellar  or  dendric-formed  small  leaves  or  seldom 
in  short  needles  (Gulewitsch  3).  On  heating  it  sublimes.  It  is  difficultly 
soluble  in  cold  water,  more  soluble  in  hot  water,  and  insoluble  in  alco- 
hol. It  behaves  like  uracil  towards  ammonia  or  baryta-water  and  silver 
nitrate.  Thymin  is  precipitated  by  phosphotungstic  acid,  which  does  not 
precipitate  uracil.  Bromine  water  is  decolorized  by  thymin,  producing 
bromthymin.  For  its  detection  we  make  use  of  the  sublimation,  the 
behavior  towards  silver  nitrate,  and  its  elementary  analysis. 

In  regard  to  the  methods  of  preparation  see  Kossel  and  Neumann 
and  W.  Jones.4 

NH— C.NHa 
Cytosin,  C4H5N30  =  CO  CH      ,    (6-amino-2-oxypyrimidine),    was     first 

N=CH 

prepared  by  Kossel  and  Steudel  from  carp-sperm,  herring  testicles,  and 
yeast  nucleic  acid ;  also  by  Levene  from  spleen  and  pancreas  nucleic  acid, 
and  finally  also  by  Wheeler  and  Johnson  from  triticonucleic  acid. 
Wheeler  and  Johnson  5  have  also  prepared  it  synthetically. 

The  free  base  is  difficultly  soluble  in  water  and  crystallizes  in  thin 
mother-of-pearl-like  leaves.  The  double  compound  with  platinum  chloride, 

1  Zeitschr.  f.  physiol.  Chem.,  37,  245. 

2  Schmiedeberg,  1.  c. ;  Arch.  f.  expt.  Path.  u.  Pharm.,  37;  Kossel  and  Neumann, 
Ber.  d.  d.  chem.  Gesellsch.,  20  and  27;  E.  Fischer  and  Roeder,  ibid.,  34. 

3  Zeitschr.  f.  physiol.  Chem.,  27. 

4  Kossel  and  Neumann,  1.  c. ;   W.  Jones,  Zeitschr.  f.  physiol.  Chem.,  29,  461. 

5  Amer.  Chem.  Journ.,  29;  see  also  foot-note  1,  page  127. 


CYTOSIN.    MINERAL  BODIES.  139 

the  crystalline  picrate,  the  nit  rale,  and  the  two  sulphates  are  of  importance 

in  the  detection  of  cytosin.    This  base  is  precipitated  by  phosphotungstic 

acid  and  by  silver  llitrate  in  the  presence  Of  an  excess  of  barium  hydroxide, 
which  is  also  of  importance  in  the  detection  of  cytosin  (KutsCHER).  Cytosin 
gives,  like  uracil,  the  murexid  reaction  with  chlorine-water  and  ammonia. 
In  regard  to  the  preparation  of  this  base,  see  Kossbl  and  Steudel  and 

Ivi    rS(  HER.1 

Mineral  Bodies.  These  bodies,  found  habitually  in  the  cells  of  higher 
plants  and  of  animals,  are  potassium,  sodium,  calcium,  magnesium,  iron, 
phosphoric  acid,  and  chlorine.  In  certain  cells  we  also  find  manganese, 
silicic  acid,  iodine,  and  also  arsenic.  We  are  chiefly  indebted  to  Liejug 
for  showing  that  the  mineral  bodies  are  as  important  for  the  normal  con- 
stitution of  the  orpins  and  tissues  as  well  as  for  the  normal  performance 
of  the  processes  of  life  as  the  organic  constituents  of  the  body.  This 
importance  of  the  mineral  constituents  follows  from  the  fact  that  we  know 
no  animal  tiOC11^  Q"d  no  animal  fluid  which  is  fre^  from  mineral  bodies, 
and  also  from  the  fact  that  certain  tissues  or  tissue  elements  contain  chiefly 
certain  mineral  bodies  and  not  others.  This  division  is,  in  general,  as  follows 
in  regard  to  the  alkali  compounds,  namely,  that  the  sodium  compounds 
occur  chiefly  in  the  fluids,  while  the  potassium  compounds  occur  especially 
in  the  form-elements.  Corresponding  to  this,  the  cells  contain  chiefly 
potassium  as  phosphate,  while  they  are  less  rich  in  sodium  and  chlorine 
compounds. 

The  importance  of  potassium  for  life  and  the  development  of  the  cell  lias 
been  shown  by  several  observations.  A  very  instructive  and  interesting 
example  of  this  action  has  been  showm  by  Loeb  2  in  his  investigations  on 
the  pathogenesis  of  the  egg  of  the  sea-annelide  Chaetopterus.  The  un- 
fertilized  egg  could,  in  sea-water  alone,  only  develop  to  the  eighth  or  six- 
teenth cell  stage;  after  a  short  stay  in  sea-wrater  to  which  KC1  was  added 
they  developed  to  the  trichophora  larva.  The  fact  that  the  Kvl  could 
not  be  replaced  by  other  chlorides  but  by  other  potassium  salts  also  shows 
that  we  are  here  dealing  with  a  specific  action  of  the  potassium  ions. 

The  importance  of  phosphoric  acid  is  not  clear;  it  is  perhaps  possible 
that  this  acid  is  important  for  the  formation  of  the  lecithins  and  nucleins, 
and  thereby  indirectly  makes  possible  the  processes  of  growth  and  division, 
which  are  dependent  upon  the  cell  nucleus.  Loew  3  has  shown,  by  means 
of  cultivation  experiments  on  algae  Spirogyra,  that  only  by  supplying 
phosphate  (in  his  experiments  potassium  phosphate)  was  the  nutrition 
of  the  cell  nucleus  made  possible,  and  thereby  the  growth  and  division  of 


1  Kossel  and  Steudel,  Zeitschr.  f.  physiol.  Chem.,  37  and  38;  Kutscher,  ibid.,  38. 

2  Amer.  Journ.  of  Physiol.,  3,  4;   Pfliiger's  Arch.,  87. 

3  Biologisches  Centralbl.,  11,  269. 


140  THE  ANIMAL  CELL. 

the  cells.  The  cells  of  the  Spirogyra  can  be  kept  alive  and  indeed  produce 
starch  and  proteids  for  some  time  without  a  supply  of  phosphates,  but 
its  growth  and  propagation  suffer. 

As  both  phosphoric  acid  and  iron  are  obtained  from  the  nuclein  sub- 
stances it  is  likely  that  these  mineral  bodies  are,  at  least  realtively,  richest 
m  the  nucleus.  As  to  the  division  of  the  mineral  bodies  between  the 
protoplasm  and  the  nucleus  we  know  nothing  with  positiveness,  and  the 
same  is  true  for  the  form  of  combination  of  the  mineral  bodies  in  the  nu- 
cleus. On  incineration  we  not  only  obtain  a  mixture  of  the  mineral  bodies 
of  the  nucleus  and  protoplasm,  but,  as  is  true  for  all  animal  fluids  and 
tissues,  the  original  relationship  is  markedly  changed.  The  combinations 
between  the  colloidal  and  mineral  substances  are  destroyed,  carbon  dioxide 
discharged,  and  sulphuric  acid  and  phosphoric  acid  may  be  produced  from 
the  organic  bodies.  The  ordinary  chemical  analysis  is  not  sufficient  for 
the  study  of  the  mineral  constituents  of  the  fluids  or  tissue,  their  forms 
of  combination  and  action;  hence  we  must  resort  to  physical-chemical 
methods. 

According  to  the  investigations  carried  on  by  these  methods  the  con- 
clusion has  been  reached,  irrespective  of  the  importance  of  the  mineral  bodies 
for  the  osmotic  tension  in  the  cells  and  tissues,  that  the  part  taken  by  the 
mineral  bodies  in  cell  life  is  essentially  the  action  of  the  ions.  For  example, 
the  permeability  of  the  blood-corpuscles,  as  well  as  other  cells  for  neu- 
tral alkali  salts,  which  wilibe  treated  in  the  following  chapter,  shows  an 
exchange  of  ions.  The  investigations  of  Maillard  on  the  toxic  action  of 
copper  salts  and  of  Paul  and  Kronig  1  of  mercury  salts,  acids,  and  alkalies 
offer  other  examples.  From  these  investigations  it  follows  that  the  tox- 
icity is  dependent  upon  the  dissociation  and  that  it  is  not  dependent  upon 
the  total  amount  of,  for  example,  copper  or  mercury  salts  present  in  the 
solution,  but  rather  upon  the  number  of  copper  or  mercury  ions. 

Beautiful  and  instructive  examples  of  the  importance  of  the  ions  for 
cell  life  have  been  shown  by  Loeb  2  and  his  collaborators.  It  is  not  within 
the  scope  of  this  book  to  give  a  detailed  account  of  this  important  work, 
but  perhaps  it  will  be  sufficient  to  give  at  least  one  example.  The  develop- 
ment of  the  eggs  of  the  fundulus  can  be  retarded  for  a  long  time  by  a  f 
normal  NaCl  solution.  On  the  addition  of  CaS04  this  retardation  is  prevented 
and  the  development  proceeds.  Other  calcium  salts  act  like  the  sulphate, 
but  alkali  sulphates  like  Na2S04  or  other  neutral  alkali  salts  do  not  have 
this  action,  hence  it  must  be  a  calcium  ion  action.  Small  quantities  of 
other  divalent  cations,  also  trivalent  ions,  act  similar  to  calcium,  while  the 

1  Maillard,  Journ.  de  Physiol,  ot  Path.,  1;  Paul  and  Kronig,  Zeitschr.  f.  physiol. 
Chem.,  12,  and  Zeitschr.  f.  Hygiene,  25. 

2  Loeb,  Amer.  Journ.  Physiol.,  3,  4,  and  «;  Pfliiger's  Arch.,  SO,  87,  88,  and  93  (with 
Gies). 


ION  ACTION.  141 

salts  of  monovalent  cations  do  not  have  this  action.  According  to  Loeb 
every  solution  which  contains  only  one  electrolyte  is  poisonous  and  this 
toxicity  can  be  prevented  by  another  electrolyte.  The  valence  determines 
the  extent  to  which  certain  ions  may  have  this  action,  because,  as  above 
Stated,  a  small  amount  of  a  divalent  ion  may  retard  the  actum  of  a  larger 
amount  of  a  monovalent  ion. 

The  chief  mass  of  the  cells  consists  of  colloids,  and  as  the  normal  func- 
tions of  the  cells  are  connected  with  a  certain  physical  condition  of  the  proto- 
plasm it  Is  natural  to  consider  the  action  of  the  ions  in  relationship  to  the 
change  in  the  state  of  the  colloids.  The  colloids  can  be  precipitated  by 
electrolytes  and  the  investigations  of  Hardy  and  Pauli  '  show  that  we 
arc  here  probably  also  dealing  with  an  ion  action.  Negatively  charged 
colloids  are,  according  to  Hardy,  precipitated  by  cations  and  positively 
charged  by  anions.  A  physiologically  balanced  salt  mixture  suitable  for 
the  normal  functions  may  also  be  produced  by  the  antagonism  of  the  ion 
action  in  a  complex  solution  containing  several  salts  (Loeb  and  Gies). 
Changes  in  one  or  the  other  direction  must  correspondingly  also  bring 
about  changes  in  the  state  of  the  colloid  by  the  action  of  the  ions.  How 
the  ions  act  in  these  instances  is  not  clear.  A  very  instructive  and  clear 
explanation  is,  however,  given  by  Hober.2 

1  Hardy,  Journ.  of  Physiol.,  24,  and  Zeitschr.  f.  physikal.  Chem.,  33;   Pauli,  Hof- 
mebster's  Beitriige,  3. 

2  Physikalische  Chemie  der  Zelle  und  der  Gewebe,  Leipzig,  1902. 


CHAPTER  VI. 
THE    BLOOD. 

The  blood  is  to  be  considered  from  a  certain  standpoint  as  a  fluid  tissue, 
and  it  consists  of  a  transparent  liquid,  the  blood-plasma,  in  which  a  vast 
number  of  solid  particles,  the  red  and  white  blood-corpuscles  (and  the  blood- 
plates)  are  suspended.  We  also  find  in  the  blood  granules  of  different  kinds, 
which  are  to  be  considered  as  transformation  products  of  the  form-ele- 
ments, j 

Outside  of  the  organism  the  blood,  as  is  well  known,  coagulates  more  or 
less  quickly;  but  this  coagulation  is  accomplished  generally  in  a  few  minutes 
after  leaving  the  body.  All  varieties  of  blood  do  not  coagulate  with  the- 
sai lie  degree  of  rapidity.  Some  coagulate  more  quickly,  others  more  slowly. 
In  vertebrates  with  nucleated  blood-corpuscles  (birds,  reptiles,  batrachiar 
and  fishes)  Delezenne  has  shown  that  the  blood  coagulates  very  slowly 
if  it  is  collected  under  precautions  so  that  it  does  not  come  in  contact  with 
the  tissues.  On  contact  with  the  tissues  or  with  tissue  extracts  they 
coagulate  in  a  few  minutes.  The  blood  with  non-nucleated  blood-corpuscles 
(mammals)  coagulates,  on  the  contrary,  very  rapidly.  The  coagulation 
of  the  blood  in  these  cases  may  also  be  somewhat  retarded  by  preventing 
the  blood  from  coming  in  contact  with  the  tissues  (Spangaro,  Arthus  2). 
Among  the  varieties  of  blood  of  mammals  thus  far  investigated  the  .blood 
of  the  horse  coagulates  most  slowly.  The  coagulation  may  be  more.,, or 
less  retarded  bv  quickly  cooling;  and  if  we  allow  equine  blood  to  flow 
lircctly  from  the  vein  into  a  glass  cylinder  which  is  not  too  wide  and  which 
has  been  cooled,  and  let  it  stand  at  0°  C,  the  blood  may  be  kept  fluid 
for  several  days.  An  upper  amber-yellow  layer  of  plasma  gradually 
separates  from  a  lower  red  layer  composed  of  blood-corpuscles  with  only 
a  little  plasma.  Between  these  is  observed  a  whitish-gray  layer  which. 
consists  of  white  blood-corpuscles. 

Thg_Blasma  thus  obtained  and  filtered  is  a  clear  amber-yellow  alkaline 
liquid  which  remains  fluid  for  some  time  when  kept  at  0°  C,  but  soon 
coagulates  at  the  ordinary  temperature. 

1  See  Latschenberger,  Wien.  Sitzungsber.,  105. 

2  Delezenne,  Compt.  rend.  Soc.  de  Biol.,  49  j  Spangaro,  Arch.  ital.  de  Biol.,  32; 
Arthus,  Journ.  d.  Physiol,  et  Pathol.,  4. 

142 


COAGULATION  OF   THE  BLOOD.  143 

The  coagulation  of  the  Mood  may  be  prevented  in  other  ways.  After 
the  injection  of  peptone  or,  more  correctly,  proteose  solutions  into  the 
blood  (.in  the  living  dog),  the  blond  docs  not  coagulate  on  leaving  the  veins 
(FANO,  Si  iimiI'T-Mi  liikim  ').  The  plasma  obtained  from  Mich  blood  by 
means  of  centrifugal  force  is  called  pepione^piasma.  According  to  Abthus 
and  HnBEB  -  the  caseoses  and  gclatoses  act  similar  to  fibrin  proteose 
in  dogs.  The  coagulation  of  the  blood  of  warm-blooded  animals  is 
prevented  by  the  injection  of  an  effusion  of  the  mouth  of  the  officinal  leech 
or  a  solution  of  the  active  substance  of  such  an  infusion,  hirudin  (Franz), 
into  the  blood  current  1 11  \v<  kaft  3).  Tf  the  blood  is  allowed  to  flow  directly, 
while  stirring  it,  into  a  neutral  salt  solution — best  a  saturated  magnesium- 
sulphate  solution  (1  vol.  salt  solution  and  3  vols,  blood) — we  obtain  a 
mixture  of  blood  and  salt  which  remains  uncoagulated  for  several  days.  The 
blood-corpuscles  which,  because  of  their  adhesiveness  and  elasticity,  would 
otherwise  pass  easily  through  the  pores  of  the  filter  paper  are  made  solid  and 
stiff  by  the  salt,  so  that  they  may  be  easily  filtered.  The  plasma  thus  ob- 
tained, which  does  not  coagulate  spontaneously,  is  called  saft-plastna. 

An  especially  good  method  of  preventing  coagulation  of  blood  consists 
in  drawing  the  Mood  into  a  dilute  solution  of  potassium  oxalate,  so  that 
the  mixture  contains  0.1  per  cent  oxalate  (Arthus  and  Paces4).  The 
soluble  calcium  salts  of  the  blood  are  precipitated  by  the  oxalate,  and 
hence  the  blood  loses  its  coagulability.  On  the  other  hand,  Horne  5  found 
that  chlorides  of  calcium,  barium,  and  strontium,  when  present  in  large 
amounts  (2-3  per  cent),  may  prevent  coagulation  for  several  days.  Accord- 
ing to  Arthus  fl  a  non-coagulable  blood-plasma  may  be  obtained  by  drawing 
the  blood  into  a  sodium  fluoride  solution  until  it  contains  0.3  per  cent  XaFl. 

On  coagulation  there  separates  in  the  previously  fluid  blood  an  insoluble 
or  a  very  difficultly  soluble  proteid  substance,  fibrin.  When  this  scpa- 
ration  takes  place  without  stirring,  the  blood  coagulates  in  a  soii 
w  1  uch,  when  carefully  severed  from  the  sides  of  the  vessel,  contracts,  and  a 
clear,  generally  yellow-colored  liquid,  the  blood-serum^  exudes.  The  solid 
coagulum  which  encloses  the  blood-corpuscles  is  called  the  blood-clot 
(placenta  sanguinis).     If  the  blood  is  beaten  during  coagulation,  the  fibrin 

arates  in  elastic  threads  or  fibrous  masses,  and  the  dejibr incited  blood 
which  separates  is  sometimes  called  cruor,7  and  consists  of  blood-corpuscles 

1  Fano,  Du  Bois-Reymond 's  Arch.,  1881;  Schmidt-Miilheim,  ibid.,  1880. 

2  Arch,  de  physiol.  (5),  8. 

3  Haycraft,  Proc.  Physiol.  Soc,  1SS4,  13,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  IS; 
Franz,  Arch.  f.  cxpt.  Path.  u.  Pharm.,  49. 

4  Archives  de  Physiol.  (5),  2,  and  Compt.  rend.,  112. 
5Journ.  of  Physiol.,  19. 

6  Joum.  de  Physiol,  et  Pharm. ,  3  and  4. 

7  The  name  cruor  is  used  in  different  senses.  We  sometimes  understand  thereby 
only  the  blood  when  coagulated  in  a  red  solid  mass,  in  other  cases  the  blood-clot  after 
the  separation  of  the  serum,  and  lastly  the  sediment  consisting  of  red  blood-corpuscles 


144  THE  BLOOD. 

and  blood-serum.  Defibrinated  blood  consists  of  blood-corpuscles  and 
serum,  while  uncoagulated  blood  consists  of  blood-corpuscles  and  blood- 
plasma.  The  essential  chemical  difference  between  blood-serum  and  blood- 
plasma  is  that  the  blood-serum  does  not  contain  even  traces  of  the  mother- 
substance  of  fibrin,  the  fibrinogen,  which  exists  in  the  blood-plasma,  and 
the  serum  is  proportionally  richer  in  another  body,  the  fibrin  ferment  (see 
page  146). 

I.    BLOOD-PLASMA  AND    BLOOD-SERUM. 

The  Blood-plasma. 

In  the  coagulation  of  the  blood  a  chemical  transformation  takes  place  in 
the  plasma.  A  part  of  the  proteids  separates  as  insoluble  fibrin.  The 
albuminous  bodies  of  the  plasma  must  therefore  be  first  described.  They 
are,  as  far  as  we  know  at  present,  fibrinogen,  nucleoproteid,  serglobulins,  and 
seralbumins. 

Fibrinogen,  which  according  to  Mathews  *  has  its  origin  in  a  destruc- 
tion of  the  leucocytes,  especially  those  from  the  intestine,  occurs  in 
blood-plasma,  chyle r  lympht  and  in  certain  transudates  and  exudates. 
It  has  the  general  properties  of  the  globulins,  but  differs  from  other 
globulins  as  follows:  In  a  moist  condition  it  forms  white  flakes  which 
are  soluble  in  dilute  common  salt  solutions,  and  which  easily  con- 
glomerate into  tough,  elastic  masses  or  lumps.  The  solution  in  NaCl 
of  5-10  per  cent  coagulates  on  heating  at  52-55°  C,  and  the  faintly 
alkaline  or  nearly  neutral  weak  salt  solution  coagulates  at  56°  C,  or  at 
exactly  the  same  temperature  at  which  the  blood-plasma  coagulates. 
Fibrinogen  solutions  are  precipitated  by  an  equal  volume  of  a  saturated 
common  salt  solution,  and  are  completely  precipitated  by  adding  an  excess 
of  NaCl  in  substance  (thus  differing  from  serglobulin) .  A  salt-free  solution 
of  fibrinogen  in  as  little  alkali  as  possible  gives  with  CaCl2  a  precipitate  con- 
taining calcium  which  soon  becomes  insoluble.  In  the  presence  of  NaCl  or 
by  the  addition  of  an  excess  of  CaCl2  the  precipitate  does  not  appear.2  It 
differs  from  myosin  of  the  muscles,  which  coagulates  at  about  the  same 
temperature,  and  from  other  proteid  bodies,  in  the  property  of  being 
converted  into  fibrin  under  certain  conditions.  Fibrinogen  has  a  strong 
decomposing  action  on  hydrogen  peroxide.  It  is  quickly  made  insoluble  by 
precipitation  with  water  or  with  dilute  acids.3  Its  specific  rotation  is 
(a)r,=  —52.5°  according  to  Mittelbach.4 

■which  is  obtained  from  defibrinated  blood  by  means  of  centrifugal  force  or  by  letting 
it  stand. 

1  Amer.  Journ.  of  Physiol.,  3. 

2  See  Hammarsten,  Zeitschr.  f.  physiol.  Cbem.,  22;  Cramer,  ibid.,  23. 

3  In  regard  to  fibrinogen  the  reader  is  referred  to  the  author's  investigations.  Pflu- 
ger's  Archiv,  19  and  22,  and  Zeitschr.  f.  physiol.  Chem.,  28. 

*  Zeitschr.  f.  physiol.  Chem.,  19. 


FIBRINOGEN  AND  FIBRIN.  14."> 

Fibrinogen  may  bo  easily  separated  from  the  salt-plasma  or  oxalate- 
plasma  by  precipitation  with  an  equal  volume  of  a  saturated  NaCl  solution. 
For  further  purification  the  precipitate  is  pressed,  redissolved  in  an  8  per 
cent  salt  solution,  the  filtrate  precipitated  by  a  saturated-salt  solution  as 
above,  and  after  precipitating  in  this  way  three  times,  the.  precipitate  at 
last  obtained  is  pressed  between  filter  paper  and  finely  divided  in  water. 

The  fibrinogen  dissolves  with  the  aid  of  the  small  amount  of  Naf'l  con- 
tained in  itself,  and  the  solution  may  be  made  salt-free  by  dialysis  with  very 
faintly  alkaline  water.  Fibrinogen  may  also,  according  to  Reye,1  be  pre- 
pared by  fractionally  precipitating  the  plasma  with  a  saturated  solution 
of  ammonium  sulphate.  We  have  no  investigations  as  regards  the  purity 
of  the  fibrinogen  so  prepared.  From  transudates  we  ordinarily  obtain 
a  fibrinogen  which  is  strongly  contaminated  with  lecithin  and  which  can 
hardly  be  purified  without  decomposing  it.  The  methods  for  the  detection 
and  quantitative  estimation  of  fibrinogen  in  a  liquid  were  formerly  based 
on  its  property  of  yielding  fibrin  on  the  addition  of  a  little  blood,  of  serum, 
or  of  fibrin  ferment.  Reye  has  suggested  the  fractional  precipitation  with 
ammonium  sulphate  as  a  quantitative  method.  The  value  of  this  method 
has  not  been  sufficiently  tested. 

Fibrinogen  stands  in  close  relation  to  its  transformation  product,. 
fibrin. 

Fibrin  is  the  name  of  that  proteid  body  which  separates  on  the  so-called 
spontaneous  coagulation  of  blood,  lymph,  and  transudates  as  well  as  in  the 
coagulation  of  a  fibrinogen  solution  after  the  addition  of  serum  or  fibrin 
ferment   (see  below). 

If  the  blood  is  beaten  during  coagulation,  the  fibrin  separates  in  elastic, 
fibrous  masses.  The  fibrin  of  the  blood-clot  may  be  beaten  to  small,  less 
elastic,  and  not  particularly  fibrous  lumps.  The  typical,  fibrous,  and 
elastic  white  fibrin,  after  washing,  stands  in  regard  to  its  solubility  close  to 
the  coagulated  proteids.  It  is  insoluble  in  water,  alcohol,  or  ether.  It 
expands  in  hydrochloric  acid  of  1  p.  m.,  as  also  in  caustic  potash  or  soda 
of  1  p.  m..  to  a  gelatinous  mass,  which  dissolves  at  the  ordinary  tempera- 
ture only  after  several  days;  but  at  the  temperature  of  the  body  it  dissolves 
more  readily  although  still  slowly.  Fibrin  may  be  dissolved  by  dilute  salt 
solutions  after  a  long  time  at  the  ordinary  temperature  or  much  more 
readily  at  40°  C,  and  this  solution  takes  place,  according  to  Arthus  and 
Huber  and  also  Dastre,2  without  the  aid  of  micro-organisms.  Accord- 
ing to  Green  and  Dastre  3  two  globulins  are  formed  in  this  solution  of 
fibrin.  Fibrin  decomposes  hydrogen  peroxide,  due  to  a  contamination  with 
catalase,  but  this  property  is  destroyed  by  heating  or  by  the  action  of 
alcohol. 

1  W.  Reye,  Uber  Nachweis  und  Bestimmung  des  Fibrinogens,  Inaug.-DLss.,  Strass- 
burg,  1898. 

2  Arthus  and  Huber,  Arch,  de  physiol.  (5),  5;  Dastre,  ibid.  (5  ,  ' . 

3  Green,  Journ.  of  Physiol.,  8;  Dastre,  1.  c. 


146  THE  BLOOD. 

What  has  been  said  of  the  solubility  of  fibrin  relates  only  to  the  typical 
fibrin  obtained  from  the  arterial  blood  of  mammals  or  man  by  whipping 
and  washing  first  with  water  and  with  common  salt  solution  and  then 
with  water  again.  The  blood  of  various  kinds  of  animals  yields  fibrin  with 
somewhat  different  properties,  and  according  to  Fermi  l  pig-fibrin  dissolves 
much  more  readily  in  hydrochloric  acid  of  5  p.  m.  than  ox-fibrin.  Fibrins 
of  varying  purity  or  originating  from  blood  from  different  parts  of  the  body 
have  unlike  solubilities. 

The  fibrin  obtained  by  beating  the  blood  and  purified  as  above  de- 
scribed is  always  contaminated  by  enclosed  blood-corpuscles  or  remains 
thereof,  and  also  by  lymphoid  cells.  It  can  only  be  obtained  pure  from 
filtered  plasma  or  filtered  transudates.  For  the  pure  preparation,  as 
well  as  for  the  quantitative  estimation  of  fibrin,  the  spontaneously  coagu- 
lating liquid  is  at  once,  or  the  non-spontaheously  coagulating  liquid 
only  after  the  addition  of  blood-serum  or  fibrin  ferment,  thoroughly 
beaten  with  a  whalebone,  and  the  separated  coagulum  is  washed  first  in 
water  and  then  with  a  5  per  cent  common  salt  solution,  and  again 
with  water,  and  finally  extracted  with  alcohol  and  ether.  If  the  fibrin  is 
allowed  to  stand  in  contact  with  the  blood  from  which  it  was  formed  for 
some  time,  it  partly  dissolves  (fibrinolysis — Dastre  2).  This  fibrinolysis 
must  be  prevented  in  the  exact  quantitative  estimation  of  fibrin  (Dastre). 

A  pure  fibrinogen  solution  may  be  kept  at  the  ordinary  temperature 
until  putrefaction  begins  without  showing  a  trace  of  fibrin  coagulation. 
But  if  to  this  solution  is  added  a  water-washed  fibrin-clot  or  a  little  blood- 
serum,,  it  immediately  coagulates  and  may  yield  perfectly  typical  fibrin. 
The  transformation  of  the  fibrinogen  into  fibrin  requires  the  presence  of 
another  body  contained  in  the  blood-clot  and  in  the  serum.  This  body, 
whose  importance  in  the  coagulation  of  fibrin  was  first  observed  by 
Buchaxax,3  was  later  rediscovered  by  Alexander  Schmidt4  and  desig- 
nated as  fibrin- ferment  or  thrombin.  The  nature  of  this  enzymotic  body  has 
not  been  ascertained  with  certainty.  Although  many  investigators, 
especially  English,  consider  fibrin-ferment  as  a  globulin,  still  more  recent 
experiments  of  Pekelharing  and  others  show  that  it  is  a  nucleoproteid 
which  according  to  Huiskamp5   occurs  in  the  thymus  gland  partly  as 

1  Zeitschr.  f.  Biologie,  28. 

2  Archives  de  Physiol.  (5),  5  and  0. 

3  London  Med.  Gazette,  1845,  617.     Cit.  by  Gamgee,  Journal  of  Physiol.,  1879. 

4  Pfliiger's  Arch.,  0;  see  also  Zur  Blutlehre,  1892,  and  Weitere  Beitrilge  zur  Blut- 
lehre,  1895. 

'■  Pekelharing,  Verharidel.  d.  Icon.  Akad.  d.  Wetensch.  te  Amsterdam,  1892,  Deel  1; 
ibid.,  1X95,  and  Centralbl.  f.  physiol.,  9;  Wright,  Proc.  Roy.  Irish  Acad.  (3),  2,  The 
Lancet,  1892,  and  On  Wooldridge's  Method,  etc.,  British  Med.  Journal,  1891;  Lilien- 
feld,  Hicmatol.  Untersuch.,  Du  Bois-Reymond's  Arch.,  1892;  Uber  Leukocytcn  und 
Blutgerinnung,  ibid.;  Halliburton  and  Brodie,  Journal  of  Physiol.,  17  and  18;  Huis- 
kamp, Zeitschr.  f.  physiol.  Chem.,  32;    Pekelharing  and  Huiskamp,  ibid.,  39. 


THROMBIN  AND  PROTHROMBIN.  1  17 

nucleohiston  and  partly  in  another  form.  Fibrin  fermont  is  produced, 
according  to  PEKELHARING,  by  the  influence  of  soluble  calcium  salts  on  a 

preformed    zymogen    existing   in    the   non-coagulated   plasma.    Schmidt 

admits  of  the  presence  of  such  a  mother-substance  of  the  fibrin  ferment 
in  the  blood  and  calls  it  prothrombin.  Thrombin  corresponds  to  other 
enzymes  in  that  the  very  smallest  amount  of  it  produces  an  action  and 
its  solution  becomes  inactive  on  heating.  The  investigations  of  1  ci.d  l  on 
the  velocity  of  coagulation  with  varying  quantities  of  thrombin  have  given 
us  a  more  positive  proof  as  to  the  enzymotic  nature  of  thrombin.  He 
found  that  at  least  within  certain  limits  an  increase  of  double  the 
quantity  of  enzyme  causes  an  increase  of  the  coagulation  velocity  to  one 
and  one  half  and  that  the  time  of  the  thrombin  action  seems  to  stand  in 
close  relationship  to  the  ride  found  by  Schutz  for  the  digestive  enzymes. 
The  optimum  of  the  thrombin  action  lies  at  about  40°  C. ;  at  70-75°  C.  the 
enzyme  is  destroyed.  The  question  as  to  whether  the  thrombin  found  in 
different  animals  is  the  same  substance  or  whether  we  find  several  throm- 
bins has  not  been  decided,  but  the  latter  is  more  probable. 

The  isolation  of  the  fibrin-ferment  has  been  tried  in  several  ways. 
Ordinarily  it  may  be  prepared  by  the  following  method  proposed  by  Alex. 
Schmidt.2  Precipitate  the  serum  or  defibrinated  blood  with  15-20  vols,  of 
alcohol  and  allow  it  to  stand  a  few  months.  The  precipitate  Is  then  filtered 
and  dried  over  sulphuric  acid.  The  ferment  may  be  extracted  from  the 
dried  powder  by  means  of  water.  Other  methods  have  been  suggested  by 
Hammarsten  and  by  Pekelharing.3 

The  preparation  of  a  thrombin  solution  as  free  as  possible  from  lime 
may  be  done  by  removing  the  lime  salts  from  the  serum  by  means  of  oxalate 
and  precipitating  the  serum  with  alcohol  and  allowing  it  to  stand  under 
alcohol  for  several  months.  The  dried  powder  is  rubbed  with  water  and 
freed  from  soluble  salts  by  repeated  lixiviation  with  water  and  the  use  of 
centrifugal  force.  Then  allow  each  gram  of  powder  to  stand  some  time  with 
100-150  c.c.  water,  filter,  and  in  this  way  obtain  a  solution  which  contains 
only  about  0.3-0.4  p.  m.  solids  and  about  0.0007  p.  m.  CaO.  (Hammarsten.) 

If  a  fibrinogen  solution  containing  salt,  as  above  prepared,  is  treated 
with  a  solution  of  fibrin-ferment,  it  coagulates  at  the  ordinary  tempera- 
ture more  or  less  quickly  and  yields  a  typical  fibrin.  Besides  the  fibrin- 
ferment  the  presence  of  neutral  salts  is  necessary,  for  without  them  Alkx. 
Schmidt  has  shown  that  fibrin  coagulation  does  not  take  place.  The  presence 
of  soluble  calcium  salts  is  not,  as  generally  admitted,  a  positive  condition 
for  the  formation  of  fibrin,  because  as  shown  by  Alex.  Schmidt.  Pekel- 
haring, and  Hammahstex,4  thrombin  can  transform  fibrinogen  into  typical 

1  Hofmeister's  Beitriige,  2. 
7  Pfliiger's  Arch.,  6. 

3  Hammarsten,  Pfliiger's  Arch.,  18;   Pekelharing,  1.  c. 

4  See  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22,  which  also  cites  the  works  of 
Schmidt  and  Pekelharing,  and  ibid.,  28. 


14S  THE   BLOOD. 

fibrin  in  the  absence  of  lime  salts  precipitable  by  oxalate.  The  fibrin  is  not 
richer  in  lime  than  the  fibrinogen  (Hammarsten)  used  to  prepare  it  if  the 
fibrinogen  and  thrombin  solution  are  employed  as  lime-free  as  possible  and 
the  view  that  the  fibrin  formation  is  connected  with  a  taking  up  of  lime  has 
been  shown  to  be  untenable.  The  quantity  of  fibrin  obtained  on  coagula- 
tion is  always  smaller  than  the  amount  of  fibrinogen  from  which  the  fibrin 
is  derived,  and  we  always  find  a  small  amount  of  protein  substance  in  the 
solution.  It  is  therefore  not  improbable  that  the  fibrin  coagulation,  in 
accordance  with  the  views  first  proposed  by  Denis,  is  a  cleavage  process  in 
which  the  soluble  fibrinogen  is  split  into  an  insoluble  proteid,  the  fibrin,, 
which  forms  the  chief  mass,  and  a  soluble  protein  substance  which  is  only 
produced  in  small  amounts.  We  find  a  globulin-like  substance  which  coagu- 
lates at  about  64°  C.  in  blood-serum  as  well  as  in  the  serum  from  coagulated 
fibrinogen  solutions.  This  substance  is  called  fibrin-globulin  by  Hammarsten. 
The  question  whether  this  substance  exists  in  the  fibrinogen  solution  as 
contamination  or  is  formed  as  a  true  cleavage  product  has  not  been  posi- 
tively decided,  and  the  question  whether  in  the  fibrinogen  coagulation  a 
cleavage  takes  place  or  not  also  requires  further  investigation.1 

There  exist  also  other  views  in  regard  to  the  processes  of  coagulation  in  the 
formation  of  fibrin  which  are  even  less  positively  founded.  The  fact  that  the 
soluble  lime  salts  are  not  necessary  for  the  transformation  of  fibrinogen  into 
fibrin  is  not  in  contradiction  to  the  other  fact  that  they  must  be  present  in 
the  coagulation  of  blood  or  plasma.  This  apparent  contradiction  may  be 
explained,  as  shown  later,  by  the  special  condition  of  the  blood-plasma,  and 
we  must  not  overlook  the  fact  that  the  coagulation  of  the  blood  is  a  much 
more  complicated  process  than  the  coagulation  of  a  fibrinogen  solution, 
inasmuch  as  the  first  involves  other  important  questions,  as,  for  instance, 
the  reason  for  the  blood  remaining  fluid  in  the  body,  the  origin  of  the 
fibrin-ferment,  and  the  importance  of  the  form-elements  in  the  coagulation, 
etc.  A  fuller  discussion  of  the  various  hypotheses  and  theories  concern- 
ing the  coagulation  of  the  blood  must  therefore  be  given  later. 

Rucleoproteid.  This  substance,  which,  as  above  mentioned,  is  considered 
by  Pekelharing  and  Huiskamp  as  identical  with  the  prothrombin  or  thrombin, 
occurs  in  the  blood-plasma  as  well  as  in  the  serum  and  is  precipitated  from  the 
latter  with  the  globulin.  It  is  similar  to  the  globulin  in  that  it  is  readily  soluble 
in  neutral  salt  solution  and  can  be  completely  salted  out  on  saturation  with 
magnesium  sulphate  and  only  separates  incompletely  on  dialysis.  It  is  much 
less  soluble  than  serglobulin  in  an  excess  of  dilute  acetic  acid  and  coagulates 
at  65-69°  C.  The  difficulty  of  solution  in  acetic  acid  is  used  by  Pekelharing 
as  an  important  means  of  separating  the  compound  proteids  from  the  globulins. 

Serglobulin,  also  called  paraglobulin  (Ivuhne),  fibrinoplaslic  substance 
(Alex.  Schmidt),  serum-casein  (Panum  2),  occur  in  the  plasma,  serum. 

1  See  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  28,  and  Heubner,  Arch.  f.  expt. 
Path.  u.  Pharm.,  49. 

2  Kiihne,  Lehrbuch  d.  physiol.  Chem.,  Leipzig,  1866-G8;  Al.  Schmidt,  Arch.  f. 
Anat.  u.  Physiol.,  1861-62;  Panum,  Virchow's  Arch.,  3  and  4. 


SERGLOBULINS.  149 

lymph,  transudates  and  exudates,  in  the  white  and  red  corpuscles,  and 
probably  in  many  animal  tissues  and  form-elements,  though  in  small  quan- 
tities.    It  is  also  found  in  the  urine  in  many  diseases. 

The  so-called  serglobulin  is  without  doubt  not  an  individual  substance,. 
but  consists  of  a  mixture  of  two  or  more  protein  bodies  which  cannot  be 
completely  and  positively  separated  from  each  other.  The  mixture  of 
globulins  obtained  from  blood-plasma  or  blood-serum  by  saturation  with 
magnesium  sulphate  or  half-saturation  with  ammonium  sulphate  consists 
of  nucleoproteid,  fibrin  globulin,  and  the  true  serglobulin  or  mixture  of 
globulins. 

The  nucleoproteid  has  already  been  discussed.  The  fibrin  globulin, 
which  occurs  in  the  serum  only  in  small  amounts,  can  be  completely  pre- 
cipitated by  XaCl.  It  has  the  general  properties  of  the  globulins,  but 
differs  from  the  serglobulins  by  a  lower  coagulation  temperature,  64- 
66°  C,  and  also  in  that  it  is  precipitated  by  (NH4)2S04  even  at  28  per  cent- 
saturation. 

If  the  globulin  obtained  by  saturation  with  magnesium  sulphate  is 
dialyzed,  then,  as  has  been  known  for  a  long  time  and  further  sub- 
stantiated by  Marcus,  only  a  part  of  the  globulin  separates  out,  while  a 
portion  remains  in  solution  and  cannot  be  precipitated  by  the  addition 
of  acid.  For  this  reason  Marcus  f  also  differentiates  between  a  water-soluble 
globulin  and  one  insoluble  in  water.  According  to  the  recent  investiga- 
tions of  Hofmeister  and  Pick  2  the  part  insoluble  in  water  corresponds 
chiefly  to  a  globulin  fraction  readily  precipitated  by  (XH4)2804  (by  28-36  vol. 
per  cent  saturated  solution)  and  the  part  soluble  in  water  corresponds 
to  a  more  difficultly  precipitable  fraction  (by  36-44  vol.  per  cent  satur- 
ated solution).  The  first  fraction  is  called  euglobulin  and  second  pseudo- 
globulin.  According  to  Forges  and  Spiro  3  the  serglobulins  can  be  sepa- 
rated by  (XHJ2S04  into  three  fractions  whose  precipitation  limits  are 
28-36,  33-42,  and  40-46  vol.  per  cent  saturated  solution.  All  three  frac- 
tions contain  globulin,  insoluble  in  water.  Freund  and  Joachim  4  have 
recently  found  that  the  euglobulin  as  well  as  the  pseudoglobulin  fraction 
is  a  mixture  of  globulin  soluble  in  water  and  globulin  insoluble  in  water 
and  consequently  the  number  of  different  globulins  in  the  serum  can  be 
still  greater. 

It  follows  from  all  these  investigations  that  either  the  difference  between 
the  globulin  soluble  in  water  and  that  insoluble  is  not  sufficient  or  that  the  frac- 
tional precipitation  with  ammonium  sulphate  is  not  suited  for  the  separation  of 
the  various  globulins.  It  must  not  be  forgotten  that  the  globulin  fractions  are 
always  contaminated  with  other  serum  constituents  and  that  they  may  influence 

1  Zeitschr.  f.  physiol.  Chem.,  28. 

2  Hofmeister 's  Beitriige,  1. 
*Ibid.,  3. 

4  Zeitschr.  f.  physiol.  Chem.,  36. 


150  THE  BLOOD. 

the  solubilities  and  precipitation  power.  As  Hammarsten  has  shown,  a  water- 
soluble  globulin  can  be  transformed  into  a  globulin  insoluble  in  water  by  careful 
purification,  and  also  the  reverse,  namely,  a  globulin  insoluble  in  water  can  some- 
times be  converted  into  one  soluble  in  water  by  allowing  it  to  lie  in  the  air.  An 
insoluble  proteid  like  casein  can  also,  according  to  Hammarsten,1  have  the  solu- 
bilities of  a  globulin  due  to  contamination  with  constituents  of  the  serum,  and 
K.  Morner  2  has  also  shown  that  a  contamination  of  the  serum  globulins  with 
soap  can  essentially  modify  the  precipitation  of  these  globulins.  Under  these 
circumstances  the  above  statements  in  regard  to  the  different  globulin  fractions 
must  be  accepted  with  great  caution. 

The  investigations  made  thus  far  upon  the  so-called  serglobulin  have 
not  led  to  any  positive  results.  That  this  globulin,  with  the  exception  of 
the  enzymes,  immune  bodies,  and  other  unknown  substances  which  are 
carried  down  by  the  various  fractions,  is  a  mixture  of  globulins  there  seems 
to  be  no  doubt.  The  serglobulin  or  the  globulin  mixture  which  is  obtained 
from  the  serum  by  the  methods  to  be  described  has  the  following  proper- 
ties. 

In  a  moist  condition  it  forms  snow-white  flaky  masses,  neither  tough 
nor  elastic,  which  always  contain  thrombin  and  hence  can  bring  about 
coagulation  in  a  fibrinogen  solution.  The  neutral  solution  is  only  incom- 
pletely precipitated  by  NaCl  added  to  saturation  and  is  not  precipitated 
by  an  equal  volume  of  a  saturated  salt  solution.  It  is  only  partly  precipi- 
tated by  dialysis  or  by  the  addition  of  acid.  On  saturation  with  mag- 
nesium sulphate  or  one  half  saturation  with  ammonium  sulphate  a  com- 
plete precipitation  is  obtained.  The  coagulation  temperature  is,  with 
5-10  per  cent  NaCl  in  solution,  69-76°,  but  more  often  75°  C.  The 
specific  rotation  of  the  solution  containing  salt  is  (o:)d=  —  47.8°  for 
the  serglobulin  from  ox-blood  (Fredericq  3).  The  various  globulin  frac- 
tions do  not  differ  essentially  from  each  other  in  their  coagulation  tem- 
peratures, specific  rotation,  refraction  coefficient  (Reiss  4),  and  their  elemen- 
tary composition.  The  average  composition  is,  according  to  Hammarsten, 
C  52.71,  H  7.01,  N  15.85,  S  1.11  per  cent.  K.  Morner5  found  1.02  per 
cent  sulphur  and  0.67  per  cent  lead-blackening  sulphur.  All  the  sulphur 
seems  to  exist  as  cystin. 

Serglobulin  contains,  as  K.  Morner  first  showed,  a  carbohydrate 
group  which  can  be  split  off.  Langstein  8  has  obtained  several  carbo- 
hydrates from  the  blood-globulin,  namely  dextrose,  laevulose,  an  animo  hexose 
not  identical  with  glucosamine,  and  probably  a  lsevorotatory  aldose  and 

1  See  Hammarsten,  Ergebnisse  d.  Physiol.,  1,  Abt.  I. 

2  Zeitschr.  f.  physiol.  Chem.,  34. 

3  Bull.  Acad.  Roy.  de  Belg.  (2),  50.  In  regard  to  paraglobulin,  see  Hammarsten, 
Pfliiger's  Arch.,  17  and  18,  and  Ergebnisse  d.  Physiol.,  1,  Abt.  I. 

*  Hofmeister's  Beitriige,  4. 
5  Zeitschr.  f.  physiol.  Chem.,  34. 

'  Morner,  Centralbl.  f.  Physiol.,  7;  Langstein,  Munch,  med.  Wochenschr.,  1902, 
1876,  and  Wien.  Sitz.-Ber.,  112,  Abt.  116,  1903. 


SERALBUMIN  S.  151 

carbohydrate  acids  of  unknown  constitution.  It  has  not  been  shown 
whether  these  small  amounts  of  carbohydrate  are  derived  from  the  globu- 
lin nr  from  other  contaminating  bodies.  According  to  Zanetti  the  blood- 
serum  contains  a  glucoproteid,  and  the  investigations  of  Eichholz  '  seem 
to  show  that  the  globulins  are  contaminated  by  a  glucoproteid. 

Serglobulin  (the  euglobulin)  may  be  easily  separated  as  a  fine  floc- 
culent  precipitate  from  blood-serum  by  neutralizing  or  making  faintly 
acid  with  acetic  acid  and  then  diluting  with  10-20  vols,  of  water.  For 
further  purification  this  precipitate  is  dissolved  in  dilute  common  salt 
solution,  or  in  water  by  the  aid  of  the  smallest  possible  amount  of  alkali, 
and  then  reprecipitated  by  diluting  with  water  or  by  the  addition  of  a 
little  acetic  acid.  All  the  serglobulin  may  also  be  separated  from  the 
serum  by  means  of  magnesium  or  ammonium  sulphate;  in  these  cases  it 
is  difficult  to  completely  remove  the  salt  by  dialysis.  As  long  as  we  are 
not  united  as  to  the  number  of  globulins  in  the  serum  it  is  not  necessary 
to  give  a  method  of  separating  the  various  globulins  in  this  mixture.  Thus 
far  the  fractional  precipitation  with  (XHJ,S04  has  been  used  chiefly.  The 
serglobulin  from  blood-serum  is  always  contaminated  by  lecithin  and 
thrombin.  A  serglobulin  free  from  thrombin  may  be  prepared  from  fer- 
ment-free transudates,  as  sometimes  from  hydrocele  fluids,  and  this  shows 
that  serglobulin  and  thrombin  are  different  bodies.  For  the  detection 
and  the  quantitative  estimation  of  serglobulin  we  may  use  the  precipi- 
tation by  magnesium  sulphate  added  to  saturation  (Hammarstex),  or  by 
an  equal  volume  of  a  saturated  neutral  ammonium-sulphate  solution  (Hof- 
meister  and  Kauder  and  Pohl  2).  In  the  quantitative  estimation  the 
precipitate  is  collected  on  a  weighed  filter,  washed  with  the  salt  solution 
employed,  dried  with  the  filter  at  about  115°  C,  then  washed  with  boiling- 
hot  water,  so  as  to  completely  remove  the  salt,  extracted  with  alcohol  and 
ether,  dried,  weighed  and  incinerated  to  determine  the  ash. 

Seralbumins  are  found  in  large  quantities  in  blood-serum,  blood-plasma. 
lymph,  transudates,  and  exudates.  Probably  they  also  occur  in  other 
animal  fluids  and  tissues.  The  proteids  which  pass  into  the  urine  under 
pathological  conditions  consist  largely  of  seralbumin. 

The  seralbumin  like  the  serglobulin  seems  also  to  be  a  mixture  of  at 
least  two  proteidbodies.  The  preparation  of  crystalline  seralbumin  (from 
horse-serum)  wasTnsTperformed  by  Gurber.  It  crystallizes  with  difficulty 
from  other  blood-sera  (Gruzewska).  Even  from  horse-serum  only  a 
portion  of  the  albumins  is  obtained  as  crystals,  and  it  is  also  possible  that 
the  amorphous  albumin,  which  is  preciptiated  by  ammonium  sulphate 
with  difficulty,  represents  two  seralbumins  (Maximowitsch).  According 
to  the  statements  of  Gurber  and  .Michel  it  seems  as  if  the  crystalline 
seralbumin  is  also  a  mixture,  but  this  is  disproved  by  reason  of  the  obser- 


1  Zanetti,  Chem.  Centralbl.,  1898,  1,  624;  Eichholz,  Journ.  of  Physiol.,  23. 
1  Hammarsten,  1.  c;  Hofmeister,  Kauder  and  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm., 
20. 


152  THE  BLOOD. 

vations  of  Schulz,  Wichmann  and  Krieger.1  We  know  nothing  as  to 
the  behavior  of  the  amorphous  fraction  of  the  seralbumin  in  this  regard. 
For  reason  of  the  different  coagulation  temperature  Halliburton  claims 
the  existence  of  three  different  albumins  in  the  blood-serum,  a  view  which, 
has  been  disputed  from  several  sides  and  recently  by  Hotjgardy.  On 
the  other  hand,  the  older  investigations  of  Kauder,  as  well  as  the  more 
recent  of  Oppenheimer,2  seem  to  indicate  a  non-unit  nature  of  the  seral- 
bumins, but  still  this  question  is  an  open  one. 

The  crystalline  seralbumin  may  perhaps  be  a  combination  with  sulphuric 
acid  (K.  Morner).  The  coagulated  albumin  obtained  from  the  aqueous 
solution  of  the  crystals  by  the  aid  of  alcohol  has  nearly  the  same  elementary 
composition  (Michel)  as  the  amorphous  mixture  of  albumin  prepared  from 
horse-serum  (Hammarsten  and  K.  Starke  3) .  The  average  composition  was 
C  53.06,  H  6.98,  N  15.99,  S  1.84  per  cent.  K.  Morner,  after  the  removal 
of  the  sulphuric  acid  from  crystalline  albumin,  found  1.73  per  cent  total 
sulphur,  which  probably  exists  only  as  cystin.  Langstein  4  has  been  able' 
to  split  off  a  nitrogenous  carbohydrate  (glucosamine)  from  crystalline  seral- 
bumin. The  quantity  was  so  small  that  the  question  is  still  undecided 
whether  or  not  the  carbohydrate  was  not  a  contamination.  The  specific 
rotation  of  crystalline  seralbumins  from  horse-serum  was  found  by  Michel, 
to  be  (a)D=  —61-61.2°  and  by  Maximowitsch  on  the  contrary  (a)D=  — 
47.47°. 

The  crystalline  and  amorphous  seralbumin  in  aqueous  solution  give 
the  ordinary  albumin  reactions.  The  coagulation  temperature  of  a  1  per 
cent  solution  poor  in  salts  is  about  50°  C,  but  rises  with  the  quantity  of 
salt.  The  coagulation  of  the  mixture  of  albumins  from  serum  generally 
takes  place  at  70-85°  C,  but  is  essentially  dependent  upon  the  reaction  and 
the  amount  of  salt  present.  Up  to  the  present  time  no  seralbumin  solution 
has  been  prepared  free  from  mineral  bodies.  A  solution  as  free  from  salts 
as  possible  does  not  coagulate  either  on  boiling  or  on  the  addition  of  alco- 
hol.    On  the  addition  of  a  little  common  salt  it  coagulates  in  both  cases.5 

Seralbumin  differs  from  the  albumin  of  the  white  of  the  hen's  egg  in 
the  following  particulars:  It  is  more  laevogyrate;  the  precipitate  formed  by 
hydrochloric  acid  easily  dissolves  in  an  excess  of  the  acid;  it  is  rendered 
less  insoluble  by  alcohol. 

1  In  regard  to  the  literature  on  the  crystalline  seralbumins,  see  Schulz:  Die  Kristal- 
lisation  von  Eiweissstoffen,  Jena,  1901  ;   Maximowitsch,  Maly's  Jahresber.,  31,  35. 

2  Halliburton,  Journ.  of  Physiol.,  5  and  7;  Hougardy.  Centralbl.  f.  Physiol.,  15, 
665;  Oppenheimer,  Verhandl.  d.  physiol.  Gesellsch. ,  Berlin,  1902. 

3  Michel,  Verhandl.  d.  phys.-med.  Gesellsch.  zu  Wi'irzburg,  29,  No.  3;  K.  Starke 
Maly's  Jahresber,  11. 

1  K.  Morner,  1.  c. ;   Langstein,  Hofmeister's  Reitriige,  1. 

s  In  regard  to  the  relationship  of  neutral  salts  to  heat  coagulation,  see  J.  Starke„ 
Sitzungsber.  d.  Gesellsch.  f.  Morph.  u.  Physiol,  in  Munchen,  1897. 


SERALBUMINS.  153 

In  preparing  the  seralbumin  mixture,  first  remove  the  globulins  accord- 
ing to  Johansson,  by  saturating  with  magnesium  sulphate  at  about  30°  C, 
and  filtering  at  the  same  temperature.  The  cooled  filtrate  is  separated 
from  the  crystallized  salt  and  is  treated  with  acetic  acid  so  that  it  contains 
about  l  per  cent.  The  precipitate  formed  is  filtered,  pressed,  dissolved 
in  water  with  the  addition  of  alkali  to  neutral  reaction  and  the  solution 
freed  from  salt  by  dialysis.  The  mixture  of  albumins  may  be  obtained 
in  a  solid  form  from  the  dialyzed  solution  by  either  evaporating  the  solu- 
tion at  a  gentle  temperature  or  by  precipitating  with  alcohol,  which  must 
be  quickly  removed.  Starke  1  has  suggested  another  method,  which  is 
also  to  be  recommended.  The  crystalline  seralbumin  may  be  prepared 
from  serum,  freed  from  globulin  by  half  saturating  with  ammonium  sul- 
phate, by  the  addition  of  more  salt  until  a  cloudiness  occurs  and  then 
proceeding  according  to  the  suggestion  of  Gurber  and  MlCHEL.  By 
acidification  with  acetic  acid  or  sulphuric  acid  the  crystallization  may 
be  considerably  enhanced.2  In  the  detection  and  quantitative  estimation 
of  seralbumin  the  filtrate  from  the  globulin  precipitated  with  magnesium 
sulphate  can  be  heated,  after  acidification  with  a  little  acetic  acid  if  neces- 
sary. The  quantity  of  seralbumin  is  best  calculated  as  the  difference 
between  the  total  proteids  and  the  globulin. 

Summary  of  the  elemental  composition  of  the  above  mentioned  and  described 
proteids  (from  horse-blood) : 

C            H           N  S  O 

Fibrinogen 52 .  93  6 .  90  16 .  66  1 .  25       22 .  26  (Hammarsten) 

Fibrin 52.68  6.83  16.91  1.10       22.48 

Fibrin-globulin 52.70  6.98  16.06       

Serglobulin 52.71  7.01  15.85  1.11       23.32               " 

Seralbumin 53. OS  7.10  15.93  1.90       21.96  (Michel) 

Langstein  3  has  also  detected  in  blood-serum  a  proteose-like  proteid 
substance  which  according  to  him  occurs  preformed  in  the  blood. 

The  Blood-serum. 

As  above  stated,  the  blood-serum  is  the  clear  liquid  which  is  pressed  ontf 
by  the  contraction  of  the  blood-clot.  It  differs  chiefly  from  the  plasma  in 
the  absence  of  fibrinogen  and  an  abundance_of  fibrin  fprmept  Considered 
qualitatively  the  blood-serum  contains  the  same  chief  constituents  as  the 
blood-plasma. 

Blood-serum  is  a  sticky  liquid  which  is  more  alkaline  towards  litmus 
than  the  plasma.  The  specific  gravity  in  man  is  1.027  to  1.032,  average 
1.02S.  The  color  is  often  strongly  or  faintly  yellow;  in  human  blood- 
serum  it  is  pale  yellow  with  a  shade  towards  green,  and  in  horses  it  is  often 
amber-yellow.    The  serum  is  ordinarily  clear;    after  a  meal  it  may  be 

Johansson,  Zeitschr.  f.  physiol.  Chera.,  9;  K.  Starke,  Maly's  Jahresber.,  11. 
'See  Hopkins  and  Pinkus,  Journ.  of  Physiol.,  23;  Krieger,  I'ber  die  Darstellung 
krystallinscher  tierischer  Eiweissstoffe,  Inaug. -Dissert,  Strassburg,  1899. 
8  Hofmeister's  Beitrage,  3. 


154  THE  BLOOD. 

opalescent,  cloudy,  or  milky  white,  according  to  the  amount  of  fat  contained 
in  the  food. 

Besides  the  above-mentioned  bodies,  the  following  constituents  are 
found  in  the  blood-plasma  or  blood-serum: 

Fat  occurs  from  1-7  p.  m.  in  fasting  animals.  After  partaking  of  food 
the  amount  is  increased  to  a  great  extent.  Soaps,  cholesterin,  and  lecithin 
are  also  found.  Cholesterin  occurs,  according  to  Hurthle,  at  least  in  part, 
as  fatty-acid  esters  (scrolin  according  to  Botjdet). 

Sugar  seems  to  be  a  physiological  constituent  of  the  plasma  and  serum. 
According  to  the  investigations  of  Abeles,  Ewald,  Kulz,  v.  Mering, 
Pavy,  Seegen,  and  Miura  2  the  sugar  found  is  dextrose.  Strauss  3  has 
also  detected  lsevulose  in  blood-serum  and  in  transudates  and  exudates. 
The  question  as  to  the  occurrence  of  other  varieties  of  sugar,  such  as  iso- 
maltose  (Pavy  and  Siau)  and  pentose  (Lepine  and  Botjlud4),  in  blood- 
serum  is  still  undecided.  Besides  sugar  the  blood-serum  contains,  as 
first  shown  by  J.  Otto,  also  another  reducing  non-fermentable  sub- 
stance. The  statements  of  Jacobsen,  Henriques,  and  Bing,5  that  this 
substance  is  jecorin  or  lecithin  sugar,  do  not  have  sufficient  foundation, 
while  the  occurrence  of  conjugated  glucuronic  acids  as  shown  by  the  in- 
vestigations of  P.  Mayer,  Lepine  and  Boulud,6  which  originate  perhaps 
from  the  form-elements  has  been  positively  shown. 

Bernard  7  has  shown  that  the  quantity  of  sugar  in  the  blood  diminishes 
more  or  less  rapidly  on  leaving  the  veins.  Lepine,  associated  with  Barral, 
has  specially  studied  this  decrease  in  the  quantity  of  sugar  and  calls  it 
glycolysis.  Lepine  and  Barral,  as  well  as  Arthus,  have  shown  that  this 
glycolysis  takes  place  in  the  complete  absence  of  micro-organisms.  It 
seems  to  be  due  to  a  soluble  glycolytic  enzyme  whose  activity  is  destroyed 
by  heating  to  54°  C.  This  enzyme  is  derived,  according  to  the  above 
investigators,  from  the  leucocytes  and  is,  according  to  Lepine,8  delivered 

1  Zeitschr.  f.  physiol.  Chem.,  21,  where  Boudet  is  also  cited.  In  regard  to  the 
quantity  of  these  esters  in  bird  serum,  see  Brown,  Amer.  Journ.  of  Physiol.,  2. 

2  See  v.  Mering,  Du  Bois-Reymond's  Archiv,  1877  (this  article  contains  numer- 
ous references);   Seegen,  Pfliiger's  Arch.,  40;   Miura,  Zeitschr.  f.  Biologie,  32. 

3  Fortschritte  d.  Media.,  1902. 

*  Pavy  and  Siau,  Journ.  of  Physiol.,  26;  Lepine  et  Boulud,  Compt.  rend.,  133,  135, 
and  136. 

"Otto,  Pfliiger's  Arch.,  35  (a  good  review  of  the  older  literature  on  sugar  in  the 
blood);  Jacobsen,  Centralbl.  f.  Physiol.,  6,  3G8;  Henriques,  Zeitschr.  f.  physiol.  Chem., 
23;   Bing,  Skand.  Arch.  f.  Physiol.,  9. 

'  Mayer,  Zeitschr.  f.  physiol.  Chem.,  32;  L6pine  et  Boulud,  1.  c. 

7  Lec;ons  sur  le  diabete.     Paris,  1877. 

8  In  regard  to  the  numerous  memoirs  of  L6pine  and  L6pine  et  Barral,  see  Lyon 
medical,  62  and  63;  Compt.  rendus,  110,  112,  113,  and  120;  Lepine,  Le  ferment  glyco- 
lytique  et  la  pathogenie  du  diabete  (Paris,  1891),  and  Revue  analytique  et  critiques 
des  travaux,  etc.,  in  Arch,  de  m6d.  exper.  (Paris,  1892);   Revue  de  mddecine,  1895; 


HEOOD  SERUM.  155 

from  the  pancreas  to  the  blood.  The  glycolysis  is,  according  to  Nasse, 
Rohmann  and  Spitzer,1  an  oxidation  which  is  produced,  according  to 
the  two  last-mentioned  investigators,  by  an  oxidation  ferment.  It  is 
surely  not  connected  with  the  survival  of  the  cells,  but  whether  it  is  a  vital 
or  a  post-mortem  process  is  not  decided.2  By  experiments  on  plasma- 
fibrin  Sieber  3  has  given  further  proof  as  to  a  glycolysis  produced  by 
enzymes  of  the  blood.  There  was  obtained  from  the  plasma-fibrin  of  some 
normal  and  some  immunized  animals  three  different  oxidation  enzymes  which 
decomposed  dextrose  by  taking  up  oxygen  and  forming  carbon  dioxide. 

The  blood-plasma  and  the  serum  as  well  as  the  lymph  also  contain 
enzymes  of  various  kinds.  According  to  Rohmann,  Bial,  Hamburger,4 
and  others,  diastases,  which  convert  starch  and  glycogen  into  maltose  or 
isomaltose,  as  well  as  a  maltoglucase  are  found  in  the  blood.  Hanriot 
has  detected  a  lipase  in  the  serum  which  decomposes  butyrin,  and  which, 
according  to  him,  decomposes  neutral  fats  and  other  esters.  The  occur- 
rence of  a  butyrinase  is  generally  admitted,  while  the  property  of  this  lipase 
of  splitting  olein  and  other  neutral  fats  is  not  generally  acknowledged 
(Arthus,  Do  yon  and  Morel  5).  This  lipolytic  property,  which  if  it  exists 
to  the  extent  that  Hanriot  ascribes  to  it,  must  not  be  confounded  with  the 
observations  first  made  by  Cohnstein  and  Michaelis  and  further  studied 
by  Weigert  6  on  the  transformation  of  fat  into  unknown  substances 
soluble  in  water.  The  occurrence  in  the  blood-serum  of  a  weak  pro- 
teolytic enzyme  whose  action  is  prevented  by  an  anti-body,  as  shown 
by  Delezexne  and  Pozerski,  has  been  confirmed  and  further  studied 
by  Hedin.7 

resides  the  above-mentioned  enzymes  and  thrombin  several  other 
enzymes  have  been  found  in  the  blood-serum,  namely,  rennin  and  trypsin, 
and  also  the  corresponding  anti-enzymes!  We  cannot  enter  into  the  dis- 
cussion  of  these,  nor  of  the  many  not  chemically  characterized  bodies  which 

Arthus,  Arch,  de  Physiol.  (5),  3,  -1;  Nasse  and  Framm,  Pfluger's  Arch.,  63;  Paderi, 
Maly's  Jahresber.,  26;  see  also  Cremer,  Physiologie  des  Glykogens  in  Ergebnisse  d. 
Physiol.,  1,  Abt.  I 

1  See  Chapter  I. 

2  See  Arthus,  1.  c;  Colenbrander,  Maly's  Jahresber.,  22;  Rywosch,  Centralbl.  f. 
Physiol.,  11,  495. 

3  Zeitschr.  f.  physiol.  Chem.,  39. 

4  Rohmann;  Rohmann  and  Hamburger,  Ber.  d.  deutsch.  chem.  Gesellsch.,  25  and 
27 ;  Pfluger's  Arch.,  52  and  60;  Bial,  Ueber  das  diast.  Ferm.,  etc.,  Inaug.-Diss.  Breslau, 
1892  (older  literature).     See  also  Pfluger's  Arch.,  52,  54,  and  55. 

s  Hanriot,  Compt.  rend.  Soc.  biol.,  48  and  54.  Compt.  rend.,  123  and  132.  Arthus, 
Journ.  de  Physiol,  et  de  Pathol.,  4;  Doyon  and  Morel,  Compt.  rend.  soc.  biol.,  54; 
Achard  and  Clerg  (Lipase  in  disease),  Compt.  rend.,  129,  and  Arch.  d.  Med.  exper.,  14. 

8  Cohnstein  and  Michaelis,  Pfluger's  Arch.,  65  and  69;   Weigert,  ibid.,  82. 

7  Delezenne  and  Pozerski,  Compt.  rend,  de  la  soc.  biolog.,  55;  Hedin,  Journ.  of 
Physiol.,  30. 


156  THE  BLOOD. 

"have  been  called  toxins  and  antitoxins,  immune  bodies,  alexins,  hcemoly- 
sins,  cytotoxics,  etc.  It  is  also  not  within  the  scope  of  this  book  to  discuss 
the  precipitins  which  can  be  used  as  a  biological  reagent  on  account  of 
their  action  upon  various  proteids.  It  may  be  sufficient  to  state  that 
the  works  of  Bordet,  Ehrlich,  Wassermann,  Schutze,  Uhlenhaut,1  and 
■others  have  shown  that  the  repeated  injection  into  an  animal  of  a  foreign 
jjroteid  body  or  of  blood  of  a  different  species  of  animal  so  changes  the 
blood  of  this  ammal  that  it  acquires  precipitating  properties  towards  the 
injected  proteid  or  the  blood.  In  this  manner  we  obtain  a  biological  reagent 
for  various  proteids  and  for  blood  of  different  animals.  This  last  behavior 
has  become  of  great  forensic  importance,  due  to  the  work  of  Uhlenhaut. 
The  various  enzymes  and  anti-enzymes,  toxins,  and  antitoxins,  precipitins 
etc.,  are  as  a  rule  precipitated  with  the  globulin,  but  differ  amongst  each 
other  by  some  being  carried  down  by  the  euglobulin,  while  the  others  are 
carried  down  by  the  pseudoglobulin  fraction. 

Among  the  bodies  which  are  found  in  the  blood,  and  without  doubt 
met  with  in  smaller  or  greater  amounts  in  the  plasma,  are  to  be  mentioned 
urea,  uric  acid  (found  in  human  blood  by  Abeles),  phosphocavnic  acid 
(Paxella  2) ,  creatine,  carbamic  acid,  paralactic  acid,  and  hippuric  acid. 
Under  pathological  conditions  the  following  bodies  have  been  found: 
Xanthine  bodies,  leucin,  tyrosin,  and  biliary  constituents. 

The  coloring-matters  of  the  blood-serum  are  very  little  known.  In 
equine  blood-serum  biliary  coloring-matters,  bilirubin,  besides  other  color- 
ing-matters, often  occur.  The  yellow  coloring-matter  of  the  serum  seems 
to  belong  to  the  group  of  luteins,  which  are  often  called  lipochromes  or  fat- 
coloring  matters.  From  ox-serum  Krtjkenberg  3  was  able  to  isolate  with 
amyl  alcohol  a  so-called  lipochrome  whose  solution  shows  two  absorption- 
bands,  of  which  one  encloses  the  line  F  and  the  other  lies  between  F  and  G. 

The  mineral  bodies  in  serum  and  plasma  are  qualitatively,  but  not 
quantitatively,  the  same.  A  part  of  the  calcium,  magnesium,  and  phos- 
phoric acid  is  removed  on  the  coagulation  of  the  fibrin.  By  means  of 
dialysis,  the  presence  of  sodium  chloride,  which  forms  the  chief  mass  or 
60-70  per  cent  of  the  total  mineral  bodies,  also  lime  salts,  sodium  car- 
bonate, besides  traces  of  sulphuric  and  phosphoric  acids  and  potassium, 
may  be  directly  shown  in  the  serum.4  Traces  of  silicic  acid,  fluorine,  cop- 
per, iron,  manganese,  and  ammonia  are  claimed  to  have  been  found  in  the 
serum.  As  in  most  animal  fluids,  the  chlorine  and  sodium  are  in  the  blood- 
serum  in  excess  of  the  phosphoric  acid  and  potassium  (the  occurrence  of 

1  The  literature  on  this  subject  may  be  found  in  bacteriological  journals  and  works. 

2  Abeles,  Wien.  med.  Jahrb.,  1887;  Panella  cited  from  Virchow's  Jahresber.  f.  1902, 
150. 

3  Sitzungsber.  d.  Jen.  Gesellsch.  f.  Med.,  1885. 

4  See  Giirber,  Verhandl.  d.  pyhs.-med   Gesellsch.  zu  Wiirzburg,  23. 


BLOOD  SEBUM.  157 

-which  in  the  serum  is  even  doubted).  The  acids  present  in  the  ash  .ire  not 
sufficient  to  saturate  the  bases  found,  a  condition  which  shows  that  a  part 
of  the  bases  is  combined  with  organic  substances,  perhaps  proteids.  This 
coincides  also  with  the  fact  that   the  great  part  of  the  alkalies  does  not 

exist  in  the  sornm  as  diffusible  alkali  compounds,  carbonate  and  phosphate, 
but  as  non-diffusible  compounds,  proteid  combinat ion.  According  to  Ham- 
burger '  37  per  cent  of  the  alkali  of  the  serum  from  horse-blood  was  dif- 
fusible and  63  per  cent  non-diffusible. 

Iodine  is  also  considered  as  a  mineral  constituent  of  the  plasma  or 
serum  because  it  seems  to  lie  habitually  found  (Gley  and  Bourcet), 
and  arsenic,  which  is  not  found  in  all  blood  but  only  in  human  blood 
(Gautier,  Bourcet  2),  is  a'iso  considered  as  a  mineral  constituent  of  the 
blood.  Iodine  occurs  to  a  greater  extent  in  human  blood  than  in  other 
bloods  and  does  not  exist  as  a  salt,  but  as  an  organic  compound 
(Bourcet). 

The  gases  of  the  blood-serum,  which  consist  chiefly  of  carbon  dioxide 
with  only  a  little  nitrogen  and  oxygen,  will  be  described  when  treating  of 
the  gases  of  the  blood. 

Because  of  the  difficulty  of  obtaining  plasma  only  a  few  analyses  have 
been  made.  As  an  example  the  results  of  the  analyses  of  the  blood-plasma 
of  the  horse  will  be  given  below.  The  analysis  No.  1  was  made  by  Hoppe- 
Seyler.3  No.  2  is  the  average  of  the  results  of  three  analyses  made  by 
Ham.marsten.     The  figures  are  given  in  1000  parts  of  the  plasma. 

Xo.  1.  No.  2. 

Water 908.4  917.6 

Solids 91.6  82.4 

Total  proteids 77.6  69.5 

Fibrin 10.1  6.5 

Globulin 3S .  4 

Seralbumin 24 . 6 

I:. I 1.21 

Extractive  substances 4.0!  ,on 

Soluble  salts 6.4  f  1J-y 

Insoluble  salts 1 .7  J 

Abderhaldex  has  made  complete  analyses  of  blood-serum  of  several 
domestic  mammals.  From  these  analyses  as  well  as  from  those  made  by 
Hammarstex  of  the  serum  from  human,  horse,  and  ox-blood  it  follows  that 
the  amount  of  solids  ordinarily  varies  between  70-97  p.  m.  The  chief  mass  of 
the  solids  consists  of  proteids,  about  55-S4  p.  m.  In  hens  Bammarstbn 
found  much  lower  values,  namely,  54  p.  m.  solids  with  only  39.5  p.  m.  proteid, 
and  Halliburton  found  only  25.4  p.  m.  proteid  in  frog's  blood.  The 
relationship  between  globulin  and  seralbumin  is,  as  shown  by  the  analya 

1  In  regard  to  method,  see  Arch.  f.  (Anat.  u.)  Physiol.,  1S9S. 

2Gley  et  Bourcet,  Compt.  rend.,  130;   Bourcet,  ibid.,  131;  Gautier,  ibid.,  131. 

3  Cit.  from  v.  Gorup-Besanez 's  Lehrbuch  der  physiol.  Chem.,  4.  Aufl.,  346. 


158  THE  BLOOD. 

Hammarsten,  Halliburton,  and  Rubbrecht,1  very  different  for  various 
animals,  but  may  also  vary  considerably  in  the  same  species  of  animal.  In 
human  blood-serum  Hammarsten  found  more  seralbumin  than  globulin,  and 
the  relationship  of  serglobulin  to  seralbumin  was  as  1:1.5.  In  regard  to 
the  quantity  of  the  remaining  organic  constituents  of  the  serum  we  refer 
the  reader  to  Abderhalden's  complete  analyses. 

The  quantity  of  mineral  bodies  in  the  serum  has  been  determined  by 
many  investigators.  The  conclusion  drawn  from  the  analyses  is  that  there 
exists  a  rather  close  correspondence  between  human  and  animal  blood- 
serum,  and  it  is  therefore  sufficient  to  give  here  the  analysis  of  C.  Schmidt  2 
of  (1)  human  blood,  and  Bunge  and  Abderhalden's  analyses  (2)  of  serum 
of  ox,  bull,  sheep,  goat,  pig,  rabbit,  dog,  and  cat.  The  results  correspond 
to  1000  parts  by  weight  of  the  serum.       1 1^^, 

1  2 

K20 0.387-0.401  0.226-0.270 

Na20 4.290-4.290  4.251-4.442 

CI 3.565-3.659  3.627-4.170 

CaO 0.15.5-0.155  0.110-0.131 

MgO 0.101 0.040-0.046 

P205  (inorg.) 0.052-0.085 

Even  if  we  bear  in  mind  that  certain  bodies,  such  as  carbon  dioxide, 
are  driven  off  during  incineration  and  that  other  bodies,  such  as  sulphuric 
acid  and  phosphoric  acid,  are  formed  from  sulphurized  and  phosphorized 
organic  substances,  still  quantitative  analyses  like  the  above  are  not 
sufficient  for  the  scientific  demands  of  to-day.  They  do  not  show  the 
true  composition  and  do  not  especially  give  an  explanation  of  the 
number  of  different  ions  present  in  the  serum  or  in  other  fluids,  a  fact 
which  is  of  the  greatest  physiological  importance.  An  answer  to  these 
questions  is  only  obtainable  by  physico-chemical  investigations,  which 
have  thus  far  been  used  in  determining  the  molecular  concentration,  the 
amount  of  electrolytes  and  non-electrolytes,  and  the  degree  of  dissociation. 

The  molecular,  or  as  Hamburger  calls  it,  the  osmotic  concentration  which  gives 
the  total  number  of  molecules  and  ions  in  the  liter,  is  measured  by  the  osmotic 

A 
pressure,  and  it  may  be  expressed  by  — —  if  we  make  use  of  the  depression  of  the 

freezing-point  (A)  instead  of  the  osmotic  pressure,  as  every  molecule  or  ion  when 
dissolved  in  1  liter  of  water  causes  a  depression  of  the  freezing-point  of  1.85°. 

The  average  depression  of  the  freezing-point  of  human  blood-serum  is 
A  =—0.526°,  and  it  seems  as  if  it  Is  a  little  lower  than  the  sera  of  other 
mammals  that  have  been  investigated:  horse  —0.560°  to  sheep  —0.619°. 
The  molecular  concentration  of  the  blood-serum  of  various  mammals  also 

1  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  25;  Hammarsten,  Pfliiger's  Arch.,  17; 
Halliburton,  Journ.  of  Physiol.,  7;  Rubbrecht,  Travaux  du  laboratoire  de  l'institut- 
de  physiologie  de  Liege,  5,  1896. 

!Cit.  from  v.  Gorup-Besanez's  Lehrbuch  der  physiol.  Chem.,  4.  Aufl.,  439. 


MOLECULAR  CONCENTRATION.  159 

differs  only  slightly  in  each  case  according  to  Bugarsky  and  Tangl1  and 
amounts  on  an  average  to  about  0.320  Mol  per  liter.  The  average 
freezing-point  depression  corresponds  closely  to  a  common  salt  solution  of 
9  p.  m.  (J  =—0.551°  to  -0.501°),  and  at  present  such  a  solution  is  con- 
sidered as  a  physiological  salt  solution  for  man  and  other  mammals. 

The  conditions  are  otherwise  with  sea  animals  which  live  in  a  medium 
rich  in  salts.  According  to  Bottazzi  the  blood  (or  the  fluid  of  the  cavities) 
of  invertebrate  sea  animals  has  an  osmotic  pressure  which  corresponds  to 
an  average  freezing-point  depression  of  J  =—2.29°,  i.e.,  exactly  the  same 
as  the  sea-water  in  which  they  live.  In  the  cartilaginous  fishes  nearly  the 
same  conditions  exist,  while  in  the  Teleostei  the  osmotic  pressure  is  much 
lower  than  the  sea-water,  but  is  about  one-half  greater  than  the  blood 
of  land  vertebrates.  The  Teleostei  are  the  first  in  the  scale  of  development 
of  animals  to  show  an  independence  of  the  osmotic  pressure  of  the  inner 
milieus  and  the  surrounding  media. 

There  is  rather  an  abundance  of  investigations  on  the  changes  in 
the  osmotic  pressure  or  the  molecular  concentration  of  the  blood-serum 
under  various  physiological  conditions  as  well  as  in  disease,  but  still  it  Is 
DO  doubt  too  early  to  draw  any  certain  conclusions  from  these  observa- 
tions. 

As  seen  from  the  above,  blood-serum  may  contain  electrolytes  as  well 
as  non-electrolytes.  Of  the  latter  the  proteids  and  also  sugar,  fat,  lecithin, 
urea,  and  the  so-called  extractive  bodies  are  of  the  greatest  importance. 
The  electrolytes  are  the  various  ions  and  the  undissociated  molecules  of 
the  salts  of  the  serum.  The  electrolytes  are  the  only  constituents  of  the 
serum  which  conduct  the  electric  current,  while  the  non-electrolytes  retard 
ine  conductivity:  The  degree  of  dissociation  can  also  be  calculated  from 
the  determination  of  the  conductivity  of  the  blood-serum. 

In  accordance  with  Hamburger  2  we  make  use  here  of  the  two  terms,  dis- 
sociation coefficient  and  dissociation  degree,  as  signifying  the  same.  This  is  not 
correct,  but  it  should  be  called  dissociation  degree  instead  of  dissociation  coefficient. 
The  dissociation  coefficient  i  is  expressed  by  the  formula  i  =  l  +  a(k—  1),  in  which 
a  represents  the  dissociation  degree  and  k  the  number  of  ions  into  which  each 
molecule  is  split. 

The  coefficient  of  dissociation  is,  according  to  Arrhexius,  the  relationship 
between  the  number  of  ions  in  a  solution  and  the  number  of  ions  which  would 
be  present  if  the  electrolytes  were  completely  dissociated.  As  the  conductivity 
of  a  solution  of  electrolytes  is  determined  by  the  number  of  ions  (admitting  that 
the  migration  velocity  of  the  ions  is  the  same  for  different  dilutions),  the  above 

Av 
coefficient  a  can  be  calculated  by  the  formula  a  =  -j — •     In  this  formula  Jv  repre- 
sents the  conductivity  of  the  original  dilution  (i.e.,  of  the  undiluted  serum)  and 

1  In  regard  to  the  literature  on  this  subject  we  refer  to  Hamburger,  "Osmoti>cher 
Druck  und  Ionenlehre,"  from  which  the  author  obtained  most  of  the  facts  given.  See 
also  Hober,  "  Physikalische  Chemie  der  Zelle  und  der  Gewebe. " 

2  Osmotischer  Druck  und  Ionenlehre,  480,  481. 


160  THE  BLOOD. 

.J  oo    the    conductivity   of    the    completely    dissociated    molecules    (ions)    after 
sufficiently  strong  dilution  of  the  serum  with  water. 

According  to  the  above  principle  the  degree  of  dissociation  of  serum 
has  been  determined  by  several  investigators,  especially  Bugarsky  and 
Tangl,  Oker-Blom,  and  Viola.  This  last  investigator  found  that  the 
dissociation  degree  of  the  blood-serum  of  healthy  human  beings  was  equal  to 
0.68-0.73.  According  to  Hamburger  the  results  thus  obtained  experi- 
mentally must  be  a  little  too  low  for  certain  reasons,  and  we  therefore  can 
consider  the  dissociation  coefficient  to  be  between  0.65  and  0.82. 

As  above  stated,  the  non  electrolytes  have  a  retarding  action  upon  the  con- 
ductivity, and  according  to  Bugarsky  and  Tangl  each  gram  of  proteid  in  100 
c.  c.  of  serum  diminishes  the  electrical  conductivity  of  the  serum  about  2.5  per 
cent.  By  making  use  of  this  fact  the  corrected  conductivity  of  the  elec- 
trolytes present  can  be  determined  from  the  conductivity.  The  corrected  con- 
ductivity is  partly  dependent  upon  the  chlorides  and  partly  upon  the  achlorides 
(which  are  nearty  identical  with  the  quantity  of  Na2C03).  If  the  amount  of 
NaCl  of  the  serum  is  determined  by  analysis  we  can  calculate  the  conductivity 
of  the  achlorides  by  subtracting  the  calculated  conductivity  of  a  solution  of 
NaCl  of  similar  concentration  (which  can  be  done  according  to  Kohlrausch's 
method)  from  the  total  corrected  conductivity.  From  these  results  we  can 
calculate  the  molecular  concentration  of  the  chlorides  and  the  achlorides.  The 
sum  of  these  two  is  subtracted  from  the  molecular  concentration  of  the  serum 
when  the  molecular  concentration  of  the  non-electrolytes  is  obtained. 

Bugarsky  and  Tangl  have  made  physico-chemical  analyses  of  blood- 
serum  of  certain  mammals  according  to  the  principle  given  above.  They 
found  that  the  molecular  concentration  was,  on  an  average,  about  0.320 
Mol  per  liter,  that  about  three-fourths  of  the  total  dissolved  molecules 
of  blood-serum  were  electrolytes,  although  the  serum  contained  about 
70-80  p.  m.  proteid  and  10  p.  m.  inorganic  bodies,  and  also  that  three- 
fourths  of  the  electrolytes  were  NaCl.  Viola  and  Bousquet  have  recorded 
less  complete  osmotic  chemical  analyses  of  blood-serum  of  diseased  and 
healthy  human  beings,  making  use  of  methods  somewhat  different  in  prin- 
ciple. 

In  the  determination  of  the  alkalinity  of  blood  and  blood-serum  up  to 
the  present  time  we  have  estimated  the  amount  of  alkali  by  titration  with 
an  acid.  "We  cannot  dispense  with  such  determinations,  although  the}r  do 
not  yield  any  information  as  to  the  true  alkalinity,  apart  from  the  fact  that 
the  results  are  dependent  upon  the  indicator  used,  because  we  understand 
as  true  alkalinity  the  concentration  of  the  hydroxy]  ions.  The  Na2C03 
is  in  aqueous  solution  more  or  less  dissociated  into  Na2+  and  COg",  depend- 
ing upon  the  dilution.  The  C03=  ions  combine  partly  with  the  H+  ions 
of  the  dissociated  water,  forming  HC03_,  and  the  corresponding  OH~  ions 
produce  the  alkaline  reaction.  If  now,  by  the  addition  of  a  little  acid,  a 
few  of  the  HO-  ioas  are  removed,  then  the  equilibrium  is  disturbed,  a  new 
quantity  of  Na2C03  is  dissociated,  and  this  process  is  repeated  every  time 


THE   RED   BLOOD-CORPUSCLES.  161 

a  now  quantity  of  acid  is  added  until  all  the  carbonate  is  dissociated.    The 

dissociation  of  the  carbonate  existing  in  the  original  concentration,  upon 
which  the  number  of  <  >H~  ions  is  dependent,  cannot  then  fore  be  determined 
by  titration.  For  these  reasons  IIoiser  has  worked  oul  a  physico-chemical 
method  of  determining  alkalinity,  based  upon  Nernst's  theory  of  liquid- 
chains.  This  method  was  used  later  by  Fabeas  and  by  Franckel  after 
a  few  changes.  The  investigations  of  these  last-mentioned  experimenters 
show  that  the  concentration  of  the  hydrox;  1  ions  in  blood-serum  and 
blood  Is  nearly  the  same  as  in  distilled  water,  and  that  these  fluids  are 
nearly  neutral  in  behavior,  which  fact  is  caused  by  the  presence  of  carbonic 
acid.  Friedenthal,1  by  testing  serum  with  phenolphthalein,  came  to 
similar  results.  Hober2  has  found  recently  by  using  his  improved  method 
that  the  concentration  of  the  hydroxy]  ions  in  the  blood  is  about  1-210-7,  or 
a  little  greater  than  in  the  purest  water.  The  quantity  of  hydroxy]  ions 
is  dependent  upon  the  carbon-dioxide  pressure  and  sinks  with  an  increased 
C02  tension.  "With  an  equally  great  COa  tension  the  normal  uncoagulated 
blood  contains  the  same  amount  of  hydroxy!  ions  as  the  defibrinated  blood. 

II.   THE   FORM-ELEMENTS   OF    THE   BLOOD. 
The  Red  Blood-Corpuscles. 

The  blood-corpnscles  axe  round,  biconcave  disks  without  membrane  and 
nucleus  in  man  and  mammalia  (with  the  exception  of  the  llama,  the  camel, 
and  their  congeners).  In  the  latter  animals,  as  also  in  birds,  amphibia,  and 
fishes  (with  the  exception  of  the  Cyclostoma) ,  the  corpuscles  have  in  general 
a  nucleus,  are  biconvex  and  more  or  less  elliptical.  The  size  varies  in 
different  animals.  In  man  thev  have  an  average  diameter  of  7  to  8  p. 
(fx= 0.001  mm.)  and  a  maximum, thieknpss  of  1.9^,  They  are  heavier. 
than  the  blood-plasma  or  serum,  and  therefore  sink  in  these  liquids.  In 
the  discharged  blood  thev  may  lie  sometimes  with  their  flat  surfaces  together, 
forming  a  cylinder  like  a  roll  of  coin  (rouleaux).  The  reason  for  this  is 
unknown,  but  as  it  may  be  observed  in  defibrinated  blood  it  seems  probable 
that  the  formation  of  fibrin  has  nothing  to  do  with  it. 

The  number  of  red  blood-corpuscles  is  different  in  the  blood  of  various 
animals.  In  the  blood  of  man  there  are  generally  5  million  red  corpuscles 
in  1  c.mm.,  and  in  woman  4  to  4.5  million. 

The  blood-corpuscles  consist  essentially  of  two  chief  constituents,,  the 
stroma,  which  forms  the  real  protoplasm,  and  the  intraglobular  contents, 
whose  cruet  constituent  is  haemoglobin.^.  We  cannot  state  anything  posi- 
tive  for  the  present  in  regard  to  a  more  detailed  arrangement  and  the 
views  on  this  subject  are  more  or  less  divergent. 

1  Hober,  Pfliiger's  Arch.,  81;    Farkas,  see  Biochem.  Centralbl.,   1,   626;  Franckel, 
Pfliiger's  Arch  ,  90;   Friedenthal,  Zeitschr.  f.  allg.  Physiol.,  1. 
;  Pfliiger's  Arch.,  89. 


162  THE  BLOOD. 

According  to  Hamburger  the  stroma  forms  a  protoplasmic  net  in  whose  meshes 
there  exists  a  red  fluid  or  semi-fluid  mass  which  consists  in  great  measure  of 
haemoglobin.  This  mass  represents  the  water-attracting  force  of  the  blood 
corpuscles,  and  besides  this  it  is  also  considered  that  the  outer  protoplasmic 
boundary  is  semi-permeable,  i.e.,  permeable  to  water  but  not  permeable  for 
certain  crystalloids.  This  view  is  hard  to  reconcile  with  the  investigations  of 
Rollett,  Stewart,  and  Oker-Blom  *  on  haemolysis  and  the  changes  in  the  elec- 
tric conductivity  of  laky  bloods.  According  to  Rollett  the  erythrocytes  consist 
of  a  hyaline  stroma  and  an  ' '  endosoma ' '  (Brucke  's  zooid)  containing  haemoglobin. 
The  haemoglobin  is  fixed  to  the  endosoma  and  the  electrolytes  to  the  stroma. 

The  red  blood- corpuscles  retain  their  volume  in  a  salt  solution  which 
has  the  same  osmotic  pressure  as  the  serum  of  the  same  blood  although 
they  may  change  their  form  in  such  solutions,  becoming  more  spherical, 
"and  may  also  undergo  a  chemical  change  (Hamburger,  Hedin,  and  others). 
Such  a  salt  solution  is  isotonic  2  with  the  blood-serum  and  its  concentra- 
tion for  a  NaCl  solution  is  approximately  9  p.  m.  for  human  and  mam- 
malian blood.  A  solution  of  greater  concentration,  a  hyperisotonic  solu- 
tion, abstracts  water  from  the  blood-corpuscles  until  osmotic  equilibrium 
is  established,  hence  the  corpuscles  shrink  and  their  volume  becomes 
smaller.  In  solutions  of  less  concentration,  hypisotonic  solutions,  the  cor- 
puscles swell  up,  due  to  the  taking  up  of  water,  and  this  swelling  may  be 
so  great,  as  on  diluting  the  blood  with  water,  that  the  haemoglobin  is  sepa- 
rated from  the  stroma  and  passes  into  the  watery  solution.  This  process 
is  called  haemolysis. 

A  haemolysis  may  also  be  brought  about  by  alternately  freezing  and 
thawing  the  blood,  as  well  as  by  the  action  of  various  chemical  substances 
which  act  as  protoplasmic  poisons.  These  bodies  are  ether,  chloroform, 
alkalies,  bile  acids,  solanin,  saponin,  and  also  the  saponin  substances,  which 
have  a  very  strong  hemolytic  action.3  Of  special  interest  in  this  regard 
are  the  hsemolysins,  which  act  like  toxins.  These  hsemolysins  may  be 
metabolic  products  of  bacteria  and  may  be  formed  by  higher  plants  and 
by  animals,  such  as  snakes,  toads,  bees,  spiders,  and  others.  Finally, 
the  hemolysins  or  globuliciclal  bodies,  occurring  normally  in  blood  sera 
or  produced  in  the  immunization  of  the  blood,  also  belong  here. 

When  the  haemoglobin  is  separated  from  the  so-called  stroma  by  a  suffi- 
ciently strong  dilution  with  water  the  stroma  is  found  in  the  solution  in  a 
swollen  condition.  By  the  action  of  carbon  dioxide,  by  the  careful  addi- 
tinn  nf  ftr.idSj  _fl,c,icl  salts,  tincture  of  iodine,  or  certain  other  bodies,  this 
residue,  rich  in  proteids,  condenses  and  in  many  cases  the  form  of  the 

'See  Hamburger,  Osmotischer  Druck  und  Ionenlehre,  1902;  Rollett,  Pfiiiger's 
Arch.,  82;  Oker-Blom.,  ibid.,  79;  Stewart,  Journ.  of  Physiol.,  24. 

2  The  work  of  Hamburger,  Hedin,  Eykman,  Koppe,  and  others  on  isotonism,  and 
the  literature  on  this  subject,  may  be  found  in  Hamburger,  Osmotischer  Druck-  und 
Ionenlehre,  1902. 

3  Koppe,  Pfiiiger's  Arch.,  99. 


THE  RED  BLOOD-CORPUSCLES.  1G3 

blood-corpuscles  may  be  again  obtained.  This  residue  has  been  called  the 
ghosts  or  stromata  of  die  red  blood-corpuscles,  ami  attempts  have  been 
made  to  isolate  it  for  chemical  investigation. 

To  Isolate  the  stromata  from  the  blood-corpuscles  they  are  washed  first 
by  diluting  the  blood  with  10-20  vols,  of  a  1-2  per  cent  common  salt 
solution  and  then  separating  the  mixture  by  centrifugal  force  or  by 
allowing  it  to  stand  at  a  low  temperature.  This  is  repeated  a  few  times 
until  the  blood-corpuscles  are  freed  from  serum.  These  purified  blood- 
corpuscles  are,  according  to  Wooldridge,  mixed  with  5-0  vols,  of  water 
and  then  a  little  ether  is  added  until  complete  solution  is  obtained.  The 
leucocytes  gradually  settle  to  the  bottom,  a  movement  which  may  b? 
accelerated  by  centrifugal  force,  and  the  liquid  which  separates  therefrom 
is  very  carefully  treated  with  a  1  per  cent  solution  of  KHS04  until  it  is 
about  as  dense  as  the  original  blood.  The  separated  stromata  are  collected 
on  a  filter  and  quickly  washed. 

Wooldridge  found  as  constituents  of  the  stromata  lecithin,  cholesterin, 
nucleoalbumin,  and  a  globulin  which,  according  to  Halliburton,  is  prob- 
ably a  nucleoproteid  which  he  calls  cell-globulin.  No  nuclein  substances 
or  seralbumin  or  albumoses  could  be  detected  by  Halliburton  and 
Friend.  The  nucleated  red  blood-corpuscles  of  the  bird  contain,  according 
to  Plosz  and  Hoppe-Seyler,1  nuclein  and  a  proteid  which  swells  to  a 
slimy  mass  in  a  10  per  cent  common  salt  solution,  and  which  seems  to  be 
closely  related  to  the  hyaline  substance  (hyaline  substance  of  Rovida,  see 
page  118)  occurring  in  the  lymph-cells.  The  non-nucleated  red  blood- 
corpuscles  are,  as  a  rule,  very  poor  in  proteid.  but  are  rich  in  haemoglobin; 
the  nucleated  corpuscles  are  richer  in  proteid  and  poorer  in  haemoglobin 
than  the  non-nucleated._ 

.  A  gelatinous,  fibrin-like  proteid  body  may  be  obtained  from  the  red 
blood-corpuscles  under  certain  cirenmstano^  This  fibrin-like  mass  has 
been  observed  on  freezing  and  then  thawing  the  sediment  of  the  blood- 
corpuscles,  or  on  discharging  the  spark  from  a  large  Levden  jar  through 
.the  blood,  or  on  dissolving  the  blood-corpuscles  of  one  kind  of  animal  in 
the  serum  of  another  (Landois,  stroma-fibrin) ;  i.e.,  in  the  so-called  hcem- 
agglutination  a  clumping  of  the  red  blood-corpuscles  into  clusters  takes 
place.  This  agglutination  can  be  brought  about  by  bodies  similar  to  the 
hsemolysins  and  also  by  serum  constituents  produced  normally  or  by 
immunization.  It  has  not  been  shown  that  a  fibrin  formation  from  the 
stroma  takes  place.  Fibrinogen  has  only  been  detected  in  the  red  cor- 
puscles of  frogs'  blood  (Alex.  Schmidt  and  Semmer  2). 

1  Wooldridge,  Du  Bois-Reymond  's  Archiv,  1SS1,  387;  Halliburton  and  Friend, 
Journal  of  Physiol.,  10;  Halliburton,  ibid.,  IS;  Plosz,  Hoppe-Seyler 's  Med.  chem. 
Untersuch.,  510. 

2  Landois,  Centralbl.  f.  d.  med.  Wissensch.,  1874,  421;  Schmidt,  Pfliiger's  Arch., 
11,  550-559. 


164  THE  BLOOD. 

Closely  related  to  the  anatomical  and  chemical  structure  of  the  erythro- 
cytes is  the  important  question  for  the  metabolism  in  the  blood  as  to  the 
permeability  of  the  erythrocytes,  that  is,  their  power  of  taking  up  substances- 
of  different  kinds.  On  this  subject  we  have  the  researches  of  Gruns,. 
Eykman,  Overton,  Koppe,  and  especially  those  of  Hamburger  and  his 
collaborators,  and  of  Hedin.1  As  a  result  of  these  researches  it  has  been 
shown  that  the  blood-corpuscles  are  completely  impermeable  for  the  ordi- 
nary varieties  of  sugar,  for  arabite  and  mannite,  and,  as  it  appears,  also  for 
the  cations  Ca++,  Sr++,  Ba++,  Mg++.  On  the  other  hand,  they  are  per- 
meable for  NH4+  ions,  as  also  for  acids  and  alkalies.  They  are  also  permeable 
for  alcohols  (more  readily  the  fewer  hydroxyl  groups  the  molecule  con- 
tains), aldehydes  (with  the  exception  of  paraldehyde),  ketones,  ethers, 
esters,  urea,  bile  salts,  and  others.  They  are  only  slightly  permeable  for 
amino  acids.  Towards  the  neutral  potassium  and  sodium  salts,  accord- 
ing to  Koppe  and  Hamburger,  the  blood-corpuscles  are  impermeable 
for  the  cations  K+  and  Na+  and  permeable,  on  the  contrary,  for  the  anions 
when  an  exchange  of  an  anion,  for  example  C03=,  in  the  blood-corpuscles 
is  possible  with  an  anion  in  the  outer  fluid,  for  example  with  CI-,  Br~r 
N03-,  etc.  Such  an  exchange  of  ions  can  be  especially  observed  according 
to  Hamburger  in  the  erythrocytes  suspended  in  NaCl  solution  and  treated 
with  C02,  when  the  outer  fluid  becomes  alkaline,  due  to  the  formation  of 
Na2C03  by  the  migration  of  CI-  ions  into  the  corpuscles  and  an  outward 
migration  of  the  C03=  ions.  For  every  one  bivalent  C03=  ion  there  must 
migrate  inward  two  univalent  Cl_  ions;  but  as  every  ion  irrespective  of 
whether  it  is  uni-  or  bivalent  has  the  same  osmotic  pressure,  therefore  the 
osmotic  pressure  of  the  blood-corpuscles  must  be  raised  and  hence  a  swell- 
ing up  takes  place,  due  to  their  taking  up  water.  The  question  as  to  how 
far  these  observations  can  be  applied  to  the  blood-corpuscles  in  their 
serum,  i.e.,  to  the  blood,  requires  further  proofs.2 

The  mineral  bodies  of  the  red  corpuscles  will  be  treated  in  connection 
with  the  quantitative  constitution  of  the  same. 

The  constituent  of  the  blood-corpuscles  existing  to  the  greatest  extent. 
is  the  red  pigment  haemoglobin. 

Blood  pigments. 

According  to  Hoppe-Seyler  the  coloring-matter  of  the  red  blood- 
corpuscles  is  not  in  a  free  state,  but  combined  with  some  other  substance.. 
TEecrvstalline  coloring-matter,  the  haemoglobin  or  oxyhemoglobin,  which 
may  be  isolated  from  the  blood,  is  considered,  according  to  Hoppe-Seyler.. 
as  a  cleavage  product  of  this  combination,  and  it  acts  in  many  ways  unlike 
the   questionable   combination   itself.    This   combination  is  insoluble  in 

1  In  regard  to  the  literature,  see  Hamburger,  Osmotischer  Druck-  und  Ionenlehre. 
3  Petry,  Hofmeister's  Beitrage,  3,  247. 


BLOOD  PIGMENTS.  165 

water  and  uncrystallizable.  It  strongly  decomposes  hydrogen  peroxide 
without  being  oxidized  itself;  it  shows  a  greater  resistance  to  certain 
chemical  reagents  (as  potassium  ferricyanide)  than  the  free  coloring-matter,, 
and  lastly  it  gives  off  its  loosely  combined  oxygen  much  more  easily  in 
vacuum  than  the  free  pigment.  To  distinguish  between  the  cleavage 
products,  the  haemoglobin  and  the  oxyhemoglobin,  Hoppe-Sevlkr  calls 
the  combination  of  the  blood-coloring  matter  of  the  venous  blood-corpuscles 
phlcbin,  and  that  of  the  arterial  artcrin.1  Since  the  above-mentioned  com- 
bination of  the  blood-coloring  matters  with  other  bodies,  for  example  (if 
they  really  do  exist)  with  lecithin,  have  not  been  closely  studied,  the  follow- 
ing statements  will  only  apply  to  the  free  pigment,  the  haemoglobin. 

The  color  of  the  blood  depends  in  part  on  hcemoglobin  and  in  part  on  a 
molecular  combination  of  this  substance  with  oxygen,  the  oxyhemoglobin. 
We  find  in  blood  after  asphyxiation  almost  exclusively  haemoglobin,  in 
arterial  blood  disproportionately  large  amounts  of  oxyhemoglobin,  and  in 
venous  blood  a  mixture  of  both.  Blood-coloring  matters  are  found  also  in 
striated  as  well  as  in  certain  smooth  muscles,  and  lastly  in  solution  in 
different  invertebrates.  The  quantity  of  haemoglobin  in  human  blood  may 
ind eed  be  somewhat  variable  under  different  circumstances,  but  amounts  to 
about  14jer  epnt  on  an  average,  or  8.5  grams,  have  been  determined  for 
each  kilo  of  the  weight  of  the  body.  Haemoglobin  belongs  to  the  group  oJ[ 
compound  proteids  and  yields  as  cleavage  products,  besides  very  small 
amounts  of  volatile  fatty  acids  and  other  bodies,  chiefly~a  proteid  globin  and 
a  coloring-matter,  hcemochromogen  (about  4  per  cent),  containing  iron. 
which  in  the  presence  of  oxygen  is  easily  oxidized  into  licematin. 

As  suggested  by  Hoppe-Seyler,  and  later  shown  by  Schuxck  and 
Marchlewski,  a  close  relationship  exists  between  chlorophyll  and  the 
blood  pigment,  because  a  derivative  of  the  first,  phylloporphyrin,  stands 
very  close  in  certain  regards  to  a  derivative  of  the  blood  pigment  haemo- 
toporphyrin.  By  the  investigations  of  Nencki  in  conjunction  with  March- 
lewski and  Zaleski  2  it  was  shown  that  haemopyrol  could  be  prepared 
from  the  derivatives  of  the  leaf  pigment  or  blood  pigments  by  reduction. 
The  fact  that  chlorophyll  and  blood  pigments  are  closely  related  and  are 
constructed  from  the  same  mother-substance  is  of  the  greatest  biological 
importance . 

The  haemoglobin  prepared  from  different  kinds  of  blood  has  not  exactly 
the  same  composition,  which  seems  to  indicate  the  presence  of   different. 

'Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  13,  479;  see  also  H.  U.  Robert,  Das 
YVirbeltierblut  in  mikro-kristallogr.  Hinsicht,  Stuttgart,  1901. 

2  Schunck  and  Marchlewski,  Annal.  d.  Chem.  u.  Pharm.,  27S,  2S4,  2SS,  290;  Nencki, 
Tier.  d.  deutsch.  chem.  Gesellsch.,  29;  Marchlewski  and  Nencki,  Ber.  d.  d.  chem. 
Gesellsch.,  34;  Nencki  and  Zaleski,  ibid.;  Marchlewski,  Chem.  Centralbl.,  1902,  I, 
1010;   Zaleski,  Zeitschr.  f.  physiol.  Chem.,  37. 


166 


THE  BLOOD. 


H 

N 

S 

Fe 

0 

P?0 

5 

7.32 

16.17 

0.390 

0  430 

21.84 

(Hoppe-Setler) 

7.22 

16.38 

0.568 

0.336 

20.93 

(Jaquet) 

6.97 

17.31 

0.650 

0.470 

19.73 

(Kossel) 

6.76 

17.94 

0.390 

0.335 

23.43 

(Zinoffsky) 

7.25 

17.70 

0.447 

0.400 

19.543 

(Hufner) 

7.38 

16.23 

0.660 

0.430 

21.360 

(Otto) 

7.38 

17.43 

0.479 

0.399 

19.602 

(Hufner) 

7.36 

16.78 

0.580 

0.480 

20.680 

(Hoppe-Seyler 

7.39 

16.09 

0.400 

0.590 

21.440 

i  c 

7.10 

16.21 

0.540 

0.430 

20.690 

6.770 

7.19 

16.45 

0.857 

0.335 

22.500 

0.197  (Jaquet) 

haemolgobins.  The  analyses  of  different  investigators  of  the  haemoglobin 
from  the  same  kind  of  blood  do  not  always  agree  with  one  another,  which 
probably  depends  upon  the  somewhat  various  methods  of  preparation. 
The  following  analyses  are  given  as  examples  of  the  constitution  of  differ- 
ent haemoglobins : 

HEemoglobin  from  the    C 

Dog 53.85 

"     54.57 

Horse 54.87 

"     51.15 

Ox 54.66 

Pig 54.17 

" 54.71 

Guinea-pig 54.12 

Squirrel 54.09 

Goose 54.26 

Hen 52.47 

The  question  whether  the  amount  of  phosphorus  in  the  haemoglobin 
from  birds  exists  as  a  contamination  or  as  a  constituent  has  not  been 
decided.  According  to  Inoko  the  haemoglobin  from  goose-blood  consists 
of  a  combination  between  nucleic  acid  and  haemoglobin.  In  the  haemo- 
globin from  the  horse  (Zinoffsky),  the  pig,  and  the  ox  (Hufner)  we  have 
1  atom  of  iron  to  2  atoms  of  sulphur,  while  in  the  haemoglobin  from  the 
dog  (Jaquet)  the  relation  is  1  to  3.  From  the  data  of  the  elementary 
analysis,  as  also  from  the  amount  of  loosely  combined  oxygen,  Hufner  * 
has  calculated  the  molecular  weight  of  dog-haemoglobin  as  14,129  and  the 
formula  C636Hl025N164FeS3O181.  According  to  the  more  recent  determina- 
tions of  Hufner  and  Jaquet  2  ox-hsemoglobin  contains  an  average  of 
0.336  per  cent  iron,  from  which  a  molecular  weight  of  16,669  may  be-  cal- 
culated. The  haemoglobin  from  various  kinds  of  blood  not  only  show  a 
diverse  constitution,  but  also  a  different  solubility  and  crystalline  form,  and  a 
varying  quantity  of  water  of  crystallization;  hence  we  infer  that  there  are 
several  kinds  of  haemoglobin.  Bohr  is  a  very  zealous  advocate  of  this 
supposition.  He  has  been  able  to  obtain  haemoglobins  from  dog-  and  horse- 
blood,  by  fractional  crystallization,  which  had  different  power  of  combining 
with  oxygen  and  containing  different  quantities  of  iron.  Hoppe-Seyler 
had  already  prepared  two  different  forms  of  haemoglobin  crystals  from 
horse-blood,  and  Bohr  concludes  from  a  collection  of  these  observations 
that  the  ordinary  haemoglobin  consists  of  a  mixture  of  different  haemo- 
globins.    In  opposition  to  this  statement  Hufner  3  has  shown  that  only 


1  Hoppe-Seyler,  Med.  chem.  Untersuch.,  370;   Jaquet,  Zeitschr.  f.  physiol.  Chem.,  >, 
14,  296;  Kossel,  ibid.,  2,  150;  Zinoffsky,  ibid.,  10;  Hufner,  Beitr.  z.  Physiol.,  Festschr. 

f.  C.  Ludwig,  1887,  74-81,  Journ.  f.  prakt.  Chem.  (N.  F.),  22;  Otto,  Zeitschr.  f.  physiol. 
Chem.,  7;  Inoko,  ibid.,  18. 

2  Arch.  f.  (Anat.  u.)  Physiol.,  1894. 

3  Bohr,   "Sur  les  combinaisons  de  l'hemoglobine    avec    l'oxygene."     Extrait  du 


OXYHEMOGLOBIN.  107 

one  haemoglobin  exists  in  ox-blood,  and  thai  this  La  probably  true  for  the 

blood  of  many  other  animals. 

Oxyhaemoglobin,  which  has  also  been  called  EUBliATOOLOBXTLIM  or 
h  T.MAiocKvsiAi.i.iN,  is  a  molecular  combination  of  haemoglobin  and  oxygen. 
For  each  molecule  of  haemoglobin  1  molecule  of  oxygen  exists;  and  the 
amount  of  loosely  combined  oxygen  which  is  united  to  1  grm.  of  haemo- 
globin (of  the  Ox)  has  been  determined  by  HtJFNEB  as  1.34  c.  C.  (calcu- 
lated at  0°  C.  and  "til)  mm.  mercury). 

According  to  Bonn,  the  facts  are  different.  He  differentiates  between  four 
different  oxyhemoglobins,  according  to  the  quantity  of  oxygen  which  they  absorb, 
namely,  a-,  ,<-,  r-,  and  o-oxyhsemoglobin,  all  having  the  same  absorption-spec- 
trum and  1  grm.  combining  with  respectively  0.4,  0.8,  1.7,  and  '2.7  c.c.  oxygen  at 
the  temperature  of  the  room  and  with  an  oxygen  pressure  of  150  mm.  mercury. 
The  p-pxyhaemoglobin  is  the  ordinary  one  obtained  by  the  customary  method  of 
preparation.  Bonn  designates  as  a-oxyhaemoglobin  the  crystalline  powder  ob- 
tained by  drying  ;  -oxyluemoglobin  in  the  air.  On  dissolving  «-oxyluemoglobin 
in  water  it  is  converted  into  ^-haemoglobin  without  decomposition,  and  the  quan- 
tity of  iron  is  increased.  On  keeping  a  solution  of  r-oxyhaemoglobin  in  a  sealed 
tube  it  is  transformed  into  ^-oxyhemoglobin,  although  the  circumstances  of  this 
change  are  not  known.  According  to  Hufneb  '  these  are  nothing  but  a  mixture  of 
genuine  and  partly  decomposed  haemoglobins. 

The  ability  of  haemoglobin  to  take  up  oxygen  seems  to  be  a  function  of 
the  iron  it  contains,  and  when  this  is  calculated  as  about  0.33-O.40  per 
cent,  then  1  atom  of  iron  in  the  haemoglobin  corresponds  to  about  2  atoms  =  1 
molecule  of  oxygen.  By  increasing  the  partial  pressure  as  well  as  by  increas- 
ing the  quantities  of  oxygen  the  haemoglobin  in  solution  takes  up  more  oxy- 
gen,  until  it  is  completely  saturated,  when  1  molecule  of  haemoglobin  is  com- 
bined with  1  molecule  of  oxygen.  Still  this  reaction  is  reversible  acc<  >r<  ling  to 
the  type  l(Hb)4-  l(02)«=±l(OHb),  and  wdth  diminished  oxygen  pressure  a  dis- 
sociation must  take  place  with  the  giving  up  of  oxygen  and  a  reformation 
of  haemoglobin.  The  equilibrium  between  oxyhaemoglobin,  haemoglobin, 
and  oxygen  is  determined  according  to  the  law  of  mass-action,  and  accord- 
ing to  the  investigations  of  Hufxer2  it  is  possible  to  calculate  the  rela- 
tionship between  oxyhaemoglobin  (OHb)  and  haemoglobin  (Hb),  at  every 
desired  partial  pressure  of  the  oxygen,  by  a  formula  suggested  by  him. 
The  dependence  of  the  reaction  between  OHb,  Hb,  and  O  upon  the  law 
ofmass-q,p.t,ion  is  naturally  of  the  very  greatest  importance  for  the  taking 
up  of  oxygen  in  the  lungsjmd  the  giving  up  of  the  same  to  the  tissues. 
It  also  makes  it  possible  to  completely  expel  the  oxygen  from  a  haemo- 
globin  solution  or  from  blood  by  means  of  a  vacuum  or  by  passing  an  indif- 
ferent gas  through  the  blood. 

Bulletin  de  l'Academie  Royale  Danoise  des  sciences,  1S90;  also  Centralbl.  f.  Physiol., 
1890,  249;  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  2;  Hufner,  Du  Bois-Reymond 'a 
Arch  ,  1894. 

•Lc. 

JArch.  f.  (Anat.  u.)  Physiol.  Abt.  Physiol.,  1901.     Supplt. 


16S  THE  BLOOD. 

Oxyhemoglobin,  which  is  generally  considered  as  a  weak  acid,  is  dextro- 
rotatory, according  to  Gam  gee.1  The  specific  rotation  for  light  of  medium 
wave-lengths  of  C  is  (o)C  =  about  +10°,  which  corresponds  also  for  carbon- 
monoxide  haemoglobin.  The  haemoglobin  is  also,  like  carbon-monoxide 
haemoglobin  (COHb)  and  methaemoglobin  (MHb),  diamagnetic,  while  the 
haematin,  which  is  richer  in  iron,  is  strongly  magnetic  (Gamgee2).  On 
passing  an  electric  current  through  an  oxyhaemoglobin  solution  the  pig- 
ment first  separates  unchanged  at  the  anode  in  a  colloidal  but  still  soluble 
form  and  is  then  gradually  transferred  to  the  cathode  in  the  colloidal 
state  (Gamgee3).  This  transportation  of  the  colloidal  haemoglobin  may 
also  be  made  to  take  place  through  an  animal  membrane  or  through  parch- 
ment paper.  According  to  Gamgee  the  haemoglobin  probably  exists  in 
such  a  colloidal  condition  in  the  blood-corpuscles. 

Oxyhaemoglobin  has  been  obtained  in  crystals  from  several  varieties  of 
blood.  These  crystals  are  blood-red,  transparent,  silky,  and  may  be  2-3  mm. 
long.  The  oxyhaemoglobin  from  squirrel's  blood  crystallizes  in  six-sided 
plates  of  the  hexagonal  system;  the  other  varieties  of  blood  }deld  needles, 
prisms,  tetrahedra,  or  plates  which  belong  to  the  rhombic  system.  The 
quantity  of  water  of  crystallization  varies  between  3-10  per  cent  for  the 
different  oxyhaemoglobins.  When  completely  dried  at  a  low  temperature 
over  sulphuric  acid  the  crystals  may  be  heated  to  110-115°  C.  without 
decomposition.  At  higher  temperatures,  somewhat  above  160°  C.,  they 
decompose,  giving  an  odor  of  burnt  horn,  and  leave,  after  complete  com- 
bustion, an  ash  consisting  of  oxide  of  iron.  The  oxyhaemoglobin  crystals 
from  difficultly  crystallizable  kinds  of  blood,  for  example,  from  such  as  ox's, 
human,  and  pig's  blood,  are  easily  soluble  in  water.  The  oxyhaemoglobins 
from  easily  crystallizable  blood,  as  from  that  of  the  horse,  dog,  squirrel, 
and  guinea-pig,  are  soluble  with  difficulty  in  the  order  above  given.  The 
oxyhaemoglobin  dissolves  more  easily  in  a  very  dilute  solution  of  alkali  car- 
bonate than  in  pure  water,  and  this  solution  may  be  kept.  The  presence 
of  a  little  too  much  alkali  causes  the  oxyhaemoglobin  to  quickly  decompose. 
The  crystals  are  insoluble  without  decolorization  in  absolute  alcohol. 
According  to  Nencki,4  it  is  hereby  converted  into  an  isomeric  or  polymeric 
modification,  called  by  him  parahcemoglobin.  Oxyhaemoglobin  is  insoluble 
in  ether,  chloroform,  benzene,  and  carbon  disulphide. 

A  solution  of  oxyhaemoglobin  in  water  is  precipitated  by  many  metallic 
salts,  but  is  not  precipitated  by  sugar  of  lead  or  basic  lead  acetate.     On 

1  Hofmeister's  Beitrage,  4. 

2  Proceedings  of  Roy.  Society,  68. 

3  Ibid.,  70. 

4  Nencki  and  Sieber,  Ber.  d.  d.  chem.  Gesellsch.,  18.  According  to  Kriiger  (see 
Biochem.  Centralbl.,  I,  40,  463),  haemoglobin  is  somewhat  changed  by  alcohol  as  well 
as  by  chloroform. 


OXYHEMOGLOBIN.  169 

heating  the  watery  solution  it  decomposes  at  about  70°  C,  and  it  splits  off 
proteid  and  h:rinatin.  It  is  also  readily  decomposed  by  acids,  alkalies, 
and  many  metallic  salts.  It  gives  the  ordinary  reactions  for  proteids,  with 
those  proteid  reagents  which  first  decompose  the  oxyhemoglobin  with 
the  splitting  ofLjuf  prot.pir^  Oxyhemoglobin  like  the  other  blood  pig- 
ments has  a  direct  oxidizing  action  upon  tincture  of  guaiacum.  It  has, 
on  the  other  hand,  like  all  blood  pigments  containing  iron,  the  property  of 
an  "ozone  transmitter"  in  that  it  turns  tincture  of  guaiacum  blue  in  the 
presence  of  reagents  containing  ozone,  such  as  old  turpentine. 

A  sufficiently  dilute  solution  of  oxyhemoglobin  or  arterial  blood  shows 
a  spectrum  with  two  absorption-bands  between  the  Fraunhofer  lines  D 
and  E.  The  one  band,  qf  which  is  narrower  but  darker  and  sharper,  lies 
on  the  line  7);  the  other,  broader,  less  defined  and  less  dark  band,  B,  lies 
at  E.  The  middle  of  the  first  band  corresponds  to  a  wave-length  x  =  578.1 
and  the  second  A=541.7.  These  bands  can  be  detected  in  a  layer  of  1  cm. 
thick  of  a  0.1  p.  m.  solution  of  oxyhemoglobin.  In  a  still  weaker  dilution 
the  band  B  first  disappears.  By  increased  concentration  of  the  solution 
the  two  bands  become  broader,  the  space  between  them  smaller  or  entirely 
obliterated,  and  at  the  same  time  the  blue  and  violet  part  of  the  spectrum 
is  darkened.  The  oxyhemoglobin  may  be  differentiated  from  other  color- 
ing matters  having  a  similar  absorption-spectrum  by  its  behavior  towards 
reducing  substances.1    (See  page  170.) 

A  great  many  methods  have  been  proposed  for  the  preparation  of 
oxyhemoglobin  crystals,  but  in  their  chief  features  they  all  agree  with 
the  following  one  suggested  by  Hoppe-Seyler:  The  washed  blood-cor- 
puscles (best  those  from  the  dog  or  the  horse)  are  stirred  with  2  vols, 
water  and  then  shaken  with  ether.  After  decanting  the  ether  and  allowing 
the  ether  which  is  retained  by  the  blood  solution  to  evaporate  in  an  open 
dish  in  the  air,  cool  the  filtered  blood  solution  to  0°  C,  add  while  stirring 
\  vol.  of  alcohol  also  cooled,  and  allow  to  stand  a  few  days  at  —5° 
to  —10°  C.  The  crystals  which  separate  may  be  repeatedly  recrystallized 
by  dissolving  in  water  of  about  35°  C,  cooling,  and  adding  cooled  alcohol  as 
above.  Lastly,  they  are  washed  with  cooled  water  containing  alcohol 
(£  vol.  alcohol)  and  dried  in  vacuum  at  0°  C.  or  a  lower  temperature.9 

For  the  preparation  of  oxyhemoglobin  crystals  in  small  quantities  from 
blood  easily  crystallized,  it  is  often  sufficient  to  stir  a  drop  of  blood  with  a 
little  water  on  a  microscope  slide  and  allow  the  mixture  to  evaporate  so  that 
the  drop  is  surrounded  by  a  dried  ring.  After  covering  with  a  cover-glass, 
the  crystals  gradually  appear  radiating  from  the  ring.    These  crystals  are 

1  Zeitschr.  f.  Biologie,  34,  contains  the  investigations  of  Gamgee  on  the  absorp- 
tion of  the  ultraviolet  rays  by  the  blood  pigment.  It  also  contains  some  of  the  earlier 
investigations. 

2  In  regard  to  the  preparation  of  oxyhemoglobin,  see  also  Hoppe-Seyler-Thier- 
f elder's  Handbuch,  7  Aufl.;  also  the  works  cited  in  foot-note  1,  page  166,  also  Schuur- 
manns-Stekhoven,  Zeitschr.  f.  physiol.  Chem.,  33,  296. 


170  THE  BLOOD. 

formed  in  a  surer  manner  if  the  blood  is  first  mixed  with  some  water  in  a 
test-tube  and  shaken  with  ether  and  a  drop  of  the  lower  deep-colored  liquid 
treated  as  above  on  the  slide. 

Haemoglobin,  also  called  reduced  haemoglobin  or  purple  cruorin 
(Stokes  x),  occurs  only  in  very  small  quantities  in  arterial  blood,  in  larger 
quantities  in  venous  blood,  and  is  nearly  the  only  blood-coloring  matter 
after  asphyxiation. 

Haemoglobin  is  much  more  soluble  than  the  oxyhemoglobin,  and  it  can 
therefore  only  be  obtained  as  crystals  with  difficulty.  These  crystals  are  as 
a  rule  isomorphous  with  the  corresponding  oxyhemoglobin  crystals,,  but  are 
darker,  having  a  shade  towards  blue  or  purple,  and  are  decidedly  more 
pleochromatic.  Its  solutions  in  water  are  darker  and  more  violet  or 
purplish  than  solutions  of  oxyhemoglobin  of  the  same  concentration. 
They  absorb  the  blue  and  the  violet  rays  of  the  spectrum  in  a  less  marked 
degree,  but  strongly  absorb  the  rays  lying  between  C-  and  D.  In  proper 
dilution  the  solution  shows  a  spectrum  with  one  broad,  not  sharply  denned 
band  between  D  and  E,  whose  darkest  part  corresponds  to  the  wave- 
length A  =  555.  This  band  does  not  lie  in  the  middle  between  D  and  E, 
but  is  towards  the  red  end  of  the  spectrum,  a  little  over  the  line  D.  \A 
hemoglobin  solutionactively  absorbs  oxygen  from  the  air  and  is  converted 
into  an  oxyhemoglobin  solution. 

A  solution  of  oxyhemoglobin  may  be  easily  converted  into  a  solution 
having  the  spectrum  of  hemoglobin  by  means  of  a  vacuum,  by  passing  an 
indifferent  gas  through  it,  or  by  the  addition  of  a  reducing  substance,  as, 
for  example,  an  ammoniacal  ferro-tartrate  solution  (Stores'  reduction 
liquid).  If  an  oxyhemoglobin  solution  or  arterial  blood  is  kept  in  a  sealed 
tube,  we  observe  a  gradual  consumption  of  oxygen  and  a  reduction  of  the 
oxyhemoglobin  into  hemoglobin.  If  the  solution  has  a  proper  concentra- 
tion, a  crystallization  of  hemoglobin  may  occur  in  the  tube  at  lower  tem- 
peratures (Hufner2). 

Pseudohaemoglobin.  Ludwig  and  Siegfried  3  have  observed  that  blood 
which  has  been  reduced  by  hyposulphites  so  completely  that  the  oxyhemoglobin 
spectrum  disappears  and  only  the  haemoglobin  spectrum  is  seen  yields  large 
amounts  of  oxygen  when  exposed  to  a  vacuum.  Blood  which  has  been  reduced 
by  the  passage  of  a  stream  of  hydrogen  through  it  until  the  oxyhemoglobin 
spectrum  disappears  acts  in  the  same  manner.  Hence  a  loose  combination  of 
hemoglobin  and  oxygen  exists  which  gives  the  hemoglobin  spectrum,  and  this 
combination  is  called  pseudohemoglobin  by  Ludwig  and  Siegfried.  Pseudo- 
hemoglobin,  whose  presence  has  been  detected  in  asphyxiation  blood  from  dogs, 
is  considered  by  Hammarsten  as  an  intermediate  step  between  hemoglobin  and 
oxyhemoglobin  on  the  reduction  of  the  latter.  The  occurrence  of  pseudohemo- 
globin does  not  seem  to  have  been  positively  proved.4 

1  Philosophical  Magazine,  28,  No.  190,  Nov.  1864. 

2  Zeitschr.  f.  physiol.  Chem.,  4. 

3  Du  Bois-Reymond's  Archiv,  1890;  see  also  Ivo  Novi,  Pfluger's  Archiv.,  56. 
*  See  Hufner,  Du  Bois-Reymond's  Arch.,  1894,  140. 


MBTHJBMOQLOBIN.  171 

Methaemoglobin.  This  name  lias  been  given  to  a  coloring-matter  which 
is  easily  obtained  from  oxyhemoglobin  as  a  transformation  product  and 
which  has  been  correspondingly  found  in  transudates  and  cystic  fluids 
containing  blood,  in  urine  in  hainaturia  or  hemoglobinuria,  also  in  urine 
and  blood  on  poisoning  with  potassium  chlorate,  amy]  nitrite  or  alkali 
nitrite,  and  many  other  bodii 

Methffimoglobin  does  not  contain  any  oxygen  in  molecular  or  dissociable 
combination,  but  still  the  oxygen  seems  to  be  of  importance  in  the  forma- 
tion of  methsemoglobin,  because  it  is  formed  from  oxyhemoglobin  in  the 
absence  of  oxygen  or  oxidizing  agents,  and  not  from  haemoglobin.  If 
arterial  blood  be  sealed  up  in  a  tube,  it  gradually  consumes  its  oxygen  and 
hpromes  vpnou^,  and  by  this  absorption  of  oxygen  a  little  m  lobin 

is  formed.  The  same  occurs  on  the  addition  of  a  small  quantity  of  acid  to 
the  blood.  By  the  spontaneous  decomposition  of  blood  some  methaemcP- 
globin  is  formed,  and  by  the  action  of  ozone,  potassium  permanganate, 
potassium  ferricyanide,  chlorates,  nitrites,  nitrobenzene,  pyrogallol,  pyro- 
catechin,  acetanilide,  and  certain  other  bodies  on  the  blood  an  abundant 
formation  of  methaemoglobin  takes  place. 

According  to  the  investigations  of  Hufxer,  Kulz,  and  Otto  1  methaemo- 
globin contains  just  as  much  oxygen  as  oxy haemoglobin,  but  it  is  more 
strongly  combined.  By  the  action  of  potassium  ferricyanide  or  potassium 
permanganate  upon  oxy  haemoglobin  first  1  molecule  oxygen  (i.e.,  the 
entire  quantity  of  loosely  combined  oxygen)  is  split  off  and  in  the  subse- 
quent methaemoglobin  formation  either  two  oxygen  atoms  (Haldane)  or 
two  hydroxy]  groups  are  combined  (Hufxer,  v.  Zeyxek  2).  Methaemo- 
globin solutions  are  reduced  to  haemoglobin  by  reducing  agents.  Jader- 
holm and  Saarbach  claim  that  methaemoglobin  is  first  converted  into 
oxyhemoglobin  and  then  into  haemoglobin  by  reducing  substances,  while 
others  (Hoppe-Seyler  and  Araki  3)   dispute  this. 

Methaemoglobin  crystallizes  as  first  shown  by  Hufxer  and  Otto  in 
brownish-red  needles,  prisms,  or  six-sided  plates.  It  dissolves  easily  in 
water;  the  solution  has  a  brown  color  and  becomes  a  beautiful  red  on  the 
addition  of  alkali.  The  solution  of  the  pure  substance  is  not  precipitated 
by  basic  lead  acetate  alone,  but  by  basic  lead  acetate  and  ammonia.  The 
absorption-spectrum  of  a  watery  or  acidified  solution  of  methaemoglobin  is, 
according  to  Jaderholm  and  Bertin-Saxs,  very  similar  to  that  of  haematin 
in  acid  solution,  but  is  easily  distinguished  from  the  latter  since,  on  the 
addition  of  a  little  alkali  and  a  reducing  substance,  the  former  passes 

1  See  Otto,  Zeitschr.  f.  physiol.  Chem.,  7. 

'Haldane,  Journ.  of  Physiol.,  22;  v.  Zeynek,  Arch.  f.  (Anat.  i.)  Physiol.,  1899; 
Hufncr,  ibid. 

3  Jaderholm,  Zeitschr.  f.  Biologie,  16;  Saarbach,  Pfliiger's  Arch,  28;  Araki,  Zeit- 
schr. f.  physiol.  Chem.,  14. 


172  THE  BLOOD. 

over  to  the  spectrum  of  reduced  haemoglobin,  while  a  haematin  solution 
under  the  same  conditions  gives  the  spectrum  of  an  alkaline  haemochromogen 
solution  (see  below).  Methsemoglobin  in  alkaline  solution  shows  two 
absorption-bands  which  are  like  the  two  oxyhemoglobin  bands,  but  they 
differ  from  these  in  that  the  band  /?  is  stronger  than  a.  By  the  side  of 
the  band  a  and  united  with  it  by  a  shadow  lies  a  third  fainter  band  between 
C  and  D,  near  to  D.  According  to  other  investigators,  Araki  and  Dit- 
trich,  a  neutral  or  faintly  acid  methsemoglobin  solution  shows  only  one 
characteristic  band,  a,  between  C  and  D,  whose  middle  corresponds  to 
about  >}  =  634.  The  two  bands  between  D  and  E  are  only  due  to  con- 
tamination with  oxyhaemoglobin  (Menzies  1). 

Crystallized  methsemoglobin  may  be  easily  obtained  by  treating  a  con- 
centrated solution  of  oxyhaemoglobin  with  a  sufficient  quantity  of  concen- 
trated postassium-ferricyanide  solution  to  give  the  mixture  a  porter-brown 
color.  After  cooling  to  0°  C.  add  \  vol.  cooled  alcohol  and  allow  the  mix- 
ture to  stand  a  few  days  in  the  cold.  The  crystals  may  be  easily  purified 
by  recrystallizing  from  water  by  the  addition  of  alcohol. 

Cyanmethaemoglobin  (cyanhsemoglobin)  is,  according  to  Haldane,  identical 
with  photomethsemoglobin  (Bock),  which  is  produced  by  the  influence  of  sunlight 
upon  a  methsemoglobin  solution  containing  potassium  ferricjranide.  It  was 
first  carefully  described  by  R.  Robert  and  obtained  in  a  crystalline  form  by 
v.  Zeynek.2  It  is  immediately  formed  in  the  cold  by  the  action  of  a  hydrocyanic- 
acid  solution  upon  methsemoglobin,  while  by  acting  upon  oxyhemoglobin  only 
at  the  body  temperature.  The  neutral  or  faintly  alkaline  solutions  show  a  spec- 
trum which  is  very  similar  to  the  hsemoglobin  spectrum. 

Acid  haemoglobin  is  a  coloring-matter  produced  by  the  action  of  very  weak 
acids  upon  oxyhemoglobin,  which  according  to  Harnack3  is  not,  as  used  to  be 
admitted,  identical  with  methsemoglobin. 

Carbon-monoxide  Hsemoglobin 4  is  the  molecular  combination  between  1 
molecule  of  hsemoglobin  and  1  molecule  of  CO,  according  to  Hufner,5  which 
contains  1.34  c.  c.  of  carbon  monoxide  (at  0°  and  760  mm.  Hg)  for  1  gram 
hsemoglobin.  This  combination  is  stronger  than  the  oxygen  combination 
of  haemoglabin.     The  oxygen  is  for  this  reason  easily  driven  out  of  oxyhsemo- 

1  Jaderholm,  1.  c. ;  Bertin-Sans,  Comp.  rend.,  106;  Dittrich,  Arch.  f.  exp.  Path.  u. 
Pharm.,  29;  Menzies,  Journ.  of  Physiol.,  17.  Important  references  on  methsemo- 
globin are  given  by  Otto,  Pfliiger's  Arch.,  31. 

2  Haldane,  Journ.  of  Physiol.,  25;  Bock,  Skand.  Arch.  f.  Physiol.,  6;  Kobert, 
Pfliiger's  Arch.,  82;  v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  33. 

1  Zeitschr.  f.  physiol.  Chem.,  26. 

*  In  reference  to  carbon-monoxide  haemoglobin  see  especially  Hoppe-Seyler,  Med. 
chem.  Untersuch.,  201;  Centralbl.  f.  d.  med.  Wissensch.,  1864  and  1865;  Zeitschr. 
f.  physiol.  Chem.,  1  and  13. 

5  Du  Bois-Reymond 's  Archiv,  Physiol.,  1894.  On  the  dissociation  constant  of 
carbon-monoxide  haemoglobin,  see  ibid.,  1895.  In  regard  to  the  contradictory  state- 
ments of  Saint-Martin  and  others  and  their  disproval,  see  Hufner,  Arch.  f.  (Anat.  u.) 
Physiol.,  1903. 


CARBON-MONOXIDE  ILEM0GL0B1N.  173 

globin  by  carbon  monoxide,  and  this  explains  the  poisonous  action  of  this 
gas.  which  kills  l>y  the  expulsion  of  the  oxygen  of  the  blood.  In  regard 
to  the  division  of  the  blood  pigments  between  the  carbon  monoxide  and 
oxygen  under  different  partial  pressures  of  both  gases  in  the  air,  we  must 
refer  to  Huknkk's  '  investigations,  whose  results  are  tabulated. 

The  carbon  monoxide  can  be  driven  out  by  a  vacuum  as  well  as  by  passing 
an  indifferent  gas  or  oxygen  or  nitric  oxide  through  the  solution  for  a  long 
time,  and  in  these  cases  haemoglobin,  oxyhemoglobin,  or  nitric-oxide 
haemoglobin  are  formed.  The  carbon  monoxide  is  also  expelled  by  po- 
tassium  ferricyanide  and  produces  methemoglobin  (Haldane2). 

Carbon-monoxide  ha?moglobin  is  formed  by  saturating  blood  or  a  hem(>_ 
globin  solution  with  carbon  monoxide,  and  may  be  obtained  as  crystals  by 
the  same  means  as  oxyluemoglobin.  These  crystals  are  isomorphous  with 
the  oxyhemoglobin  crystals,  but  are  less  soluble  and  more  stable,  and  their 
bluish-red  color  is  more  marked.  For  the  dpfry.tinn  nf  carbon-monoxide 
hemoglobin  its  absorption-spectrum  is  of  the  greatest  importance.  This 
spectrum  shows  two  hands  whjeh_a  re  very  similar  to  those  of  oxyhemoglobin, 
but  they  occur  more  towards  the  violet  part  of  the  spectrum.  The  middle 
of  the  first  band  corresponds  to  /--.">72  and  the  second  to  /-  o.'lb.  These 
bands  do  not  change  noticeably  on  the  addition  of  reducing  substances ;  this 
constitutes  an  important  difference  between  carbon-monoxide  haemoglobin 
and  oxyhemoglobin.  If  the  blood  contains  oxyhemoglobin  and  carbon- 
monoxide  hemoglobin  at  the  same  time,  we  obtain  on  the  addition  of  a 
reducing  substance  (ammoniacal  ferro-tartrate  solution)  a  mixed  spectrum 
originating  from  the  hemoglobin  and  carbon-monoxide  hemoglobin. 

A  great  many  reactions  have  been  suggested  for  the  detection  of  carbon- 
monoxide  hemoglobin  in  medico-legal  cases.  A  simple  and  at  the  same 
time  a  good  one  is  Hoppe-Seyler's  soda  test.  The  blood  is  treated  with 
double  its  volume  of  caustic-soda  solution  of  1.3  sp.  gr.,  by  which  ordinary 
blood  is  converted  into  a  dingy  brownish  mass,  which  when  spread  out 
on  porcelain  is  brown  with  a  shade  of  green.  Carbon-monoxide  blood 
gives  under  the  same  conditions  a  red  mass,  which  if  spread  out  on  porce; 
lain  shows  a  beautiful  red  color.  Several  modifications  of  this  test  have 
been  proposed.  Another  very  good  reagent  is  tannic  acid,  which  gives 
with  dilute  normal  blood  a  brownish-green  precipitate  and  with  carbon- 
monoxide  blood  a  pale  crimson-red  precipitate.3 

As  according  to  Bohr  there  are  several  oxyhemoglobins,  so  also,  according  to 
Bohr  and  Bock,4  there  are  several  carbon-monoxide  haemoglobins,  with  different 

1  Arch.  f.  expt.  Path.  u.  Pharm.,  48. 

2  Journ.  of  Physiol.,  22. 

3  In  regard  to  this  test  (as  suggested  by  Kunkel)  and  others  we  refer  to  Kostin, 
Pfluger's  Arch.,  84,  which  contains  a  very  excellent  summary  of  the  literature  on  the 
subject. 

'Centralbl.  f.  Physiol.,  S,  and  Maly's  Jahresber.,  25. 


174  THE  BLOOD. 

amounts  of  carbon  monoxide.  As  haemoglobin  can  unite  with  oxygen  and  carbon: 
dioxide  simultaneously,  as  shown  by  Bohr  and  Torup,  so  also  can  it  unite  with 
carbon  monoxide  and  carbon  dioxide  simultaneously  independently  of  each  other. 
Carbon-monoxide  methaemoglobin  has  been  prepared  by  Weil  and  v.  Anrep 
by  the  action  of  potassium  permanganate  on  carbon-monoxide  haemoglobin, 
but  this  is  contradicted  by  Bertin-Sans  and  Moitessier.1  Sulphur  methaemo- 
globin is  the  name  given  by  Hoppe-Seyler  to  that  coloring  matter  which  is 
formed  by  the  action  of  sulphuretted  hydrogen  upon  oxyhemoglobin.  The 
solution  has  a  greenish-red,  dirty  color,  and  shows  two  absorption-bands  between 
C  and  D.  This  coloring  matter  is  claimed  to  be  the  greenish  color  seen  on  the 
surface  of  putrefying  flesh.  According  to  Harnack2  the  conditions  are  different 
when  H2S  is  passed  through  an  oxygen-free  solution  of  haemoglobin  (or  carbon- 
monoxide  hemoglobin) .  The  sulf haemoglobin  thus  formed  shows  one  band  in 
the  red  between  C  and  D. 

Carbon-dioxide  Haemoglobin,  Carbohccmoglobin^  Haemoglobin,  accord- 
ing to  Bohr  and  Torup,3  also  forms  a  molecular  combination  with  carbon 
dioxide  whose  spectrum  is  similar  to  that  of  haemoglobin.  According  to 
Bohr  there  are  three  different  carbohsemoglobins,  namely,  a-,  /?-,  and 
7--carbohsemoglobin,  in  which  1  grm.  combines  with  respectively  1.5,  3,  and 
6  c.  c.  C02  (measured  at  0°  C.  and  760  mm.)  at  18°  C.  and  a  pressure  of  60 
mm.  mercury.  If  a  haemoglobin  solution  is  shaken  with  a  mixture  of  oxygen 
and  carbon  dioxide,  the  haemoglobin  combines  loosely  with  the  oxygen 
as  well  as  with  the  carbon  dioxider  independently  of  each  other,  just  as  if 
each  gas  existed  alone  (Bohr).  He  considers  that  the  two  gases  are  com- 
bined with  different  parts  of  the  haemoglobin,  namely,  the  oxygen  with  the 
pigment  nucleus  and  the  carbon  dioxide  with  the  proteid  component. 
According  to  Torup  the  haemoglobin  must  therefore  be  partly  decomposed 
by  the  carbon  dioxide  setting  free  some  proteid. 

Nitric-oxide  Haemoglobin  is  also  a  crystalline  molecular  combination 
which  is  even  stronger  than  the  carbon-monoxide  haemoglobin.  Its  solution 
shows  two  absorption-bands  which  arc  paler  and  less  sharp  than  the  carbon- 
monoxide  haemoglobin  bands,  and  they  do  not  disappear  on  the  addition  of 
reducing  bodies.  Haemoglobin  also  forms  a  molecular  combination  with 
acetylene. 

Hasmorrhodin  is  the  name  given  by  Lehmann  to  a  beautiful  red  pigment 
soluble  in  alcohol  and  ether  and  which  is  extracted  from  meat  and  meat  products 
by  boiling  alcohol  and  which  seems  to  be  produced  by  the  action  of  small  amounts  of 
nitrites.  Another  pigment  isolated  by  Lewin1  from  the  blood  of  animals  poi- 
soned by  phenylhydrazine  has  been  called  hcemoverdin.  By  heating  a  solu- 
tion of  blood  pigment  treated  with  caustic  potash  and  mixed  with  alcohol  to 
60°  C.  we  obtain,  according  to  v.  Klaveren,  a  pigment  which  he  calls  kathcemo- 

1  v.  Anrep,  Du  Bois-Reymond's  Arch.,  1880;  Sans  and  Moitessier,  Compt.  rend.,  113. 

2  Med.-chem.  Untersuch.,  151.  See  Araki,  Zeitschr.  f.  physiol.  Chem.,  14;  Har- 
nack, 1.  c. 

3  Bohr,  Extrait  du  Bull,  de  TAcad.  Danoise,  1890;  Centralbl.  f.  Physiol.,  4.; 
Torup,  Maly's  Jahresber.,  17. 

4  K.  B.  Lehmann,  Sitzungsber.  d.  phys.-med.  Gesellsch.  Wiirzburg,  1899;  Lewin, 
Compt.  rend.,  133. 


DECOMPOSITION   l'UobUCTS  OF   THE  BLOOD  PIGMENTS.       175 

gloibin,  but  called  by  Abnold,1  who  first  obtained  it,  neutral  hamatin,  which  is  pro- 
duced by  the  splitting  off  of  a  ferruginous  complex.  This  pigment  still  contains 
proteid  but  is  poorer  in  iron  than  the  haemoglobin  or  methtemoglobin  and  proba- 
bly forms  an  intermediary  product  in  the  conversion  of  the  above  into  haunatin. 

Decpmposition  products  of  the  blood  yigments.  By  its  decomposition 
haemoglobin  yields,  as  previously  stated,  a  prokid,  which  has  been  called 
yloliiji  (Vhkyvm,  Schulz),  and  a  ferruginous  piyment  us  chief  products. 
According  to  LiAWBOW  (.)4.()'.)  per  cent  proteid,  4.47  per  cent  ha-matin,  and 
1.44  per  cent  other  bodies  are  produced  in  this  decomposition.  The  globin, 
which  was  isolated  and  studied  by  Schulz,2  differs  from  most  other  pro- 
teids  by  containing  a  high  amount  of  carbon,  54.97  per  cent,  with  16.89 
per  cent  of  nitrogen.  It  is  insoluble  in  water  but  very  easily  soluble  in  acids 
or  alkalies.  It  is  not  dissolved  by  ammonia  in  the  presence  of  ammonium 
chloride.  Nitric  acid  precipitates  it  in  the  cold  but  not  when  warm.  It 
may  be  coagulated  by  heat,  but  the  coagulum  is  readily  soluble  in  acids. 
Because  of  these  reactions  it  is  considered  as  a  histon  by  Schulz. 

The  pigment  split  off  is  different,  depending  upon  the  conditions  under 
which  the  cleavage  takes  place.  If  the  decomposition  takes  place  in  the 
absence  of  oxygen,  a  coloring  matter  is  obtained  which  is  called  by  Ho ppe- 
Sey j ,er  faprwchrornngpT^  hy  other  investigators  (Stokes)  reduced  hoematin. 
In  the  presence  of  oxygen,  haemochromogen  is  quickly  oxidized  to  haematin, 
and  there  is  therefore  obtained  in  this  case  hcematin  as  a  colored  decomposi- 
tion product.  As  riaemochrrnringpn  is  Aasily  converged  by  oxygen  into- 
haematin,  so  this  latter  may  be  reconverted  into  haemochromogen  by  ret  1  u<  •- 
lng  substances.  _ 

Haemochromogen  was  discovered  by  Hoppe-Seyler.3  Haemochromogen 
is,  according  to  Hoppe-Seyler,  the  colored  atomic  group  of  haemoglobin 
and  its  combination  with  gases,  and  this  atomic  group  is  combined  with 
proteids  in  the  pigment.  The  characteristic  absorption  of  light  depends 
on  the  haemochromogen,  and  it  is  also  this  atomic  group  which  binds  in  the 
oxyhemoglobin  1  molecule  of  oxygen  and  in  the  carbon-monoxide  haemo- 
globin 1  molecule  of  carbon  monoxide  with  1  atom  of  iron.  Hoppe-Seyler 
has  observed  a  combination  between  haemochromogen  and  carbon  mon- 
oxide, and  this  combination  shows  the  spectral  appearance  of  carbon- 
monoxide  haemoglobin.  By  the  reduction  of  haematin  in  alcoholic  ammo- 
niacal  solution  by  means  of  hydrazine  v.  Zeyxek  '  was  able  to  obtain  the 
solid  brownish-red  ammonia  combination. 

An  alkaline  haemochromogen  solution  has  a  beautiful  cherry-red  color. 
It  shows  two  absorption-bands,  first  described  by  Stokes,  one  of  which. 

1  v.  Klaveren,  Zeitschr.  f.  physiol.  Chem.,  33;  Arnold,  ibid.,  29. 
'  Lawrow,  ibid.,  26;  Schulz,  ibid.,  24. 
3  Hoppe-Seyler,  ibid.,  13. 
'Ibid.,  25.  " 


176  THE  BLOOD. 

is  ^ark  and  whose  center  corresponds  to  A=556.4  between  D  and  E, 
and  a  second  broader  band,  less  dark,  which  covers  the  Fraunhofer 
lines  E  and  b.  The  middle  of  this  band  corresponds  to  X  =  520.4.  In  acid 
solution  hsemochromogen  shows  four  bands,  which,  according  to  Jader- 
holm,1  depend  on  a  mixture  of  hsemochromogen  and  haematoporphyrin 
(see  below),  this  last  formed  by  a  partial  decomposition  resulting  from 
the  action  of  the  acid. 

Hsemochromogen  may  be  obtained  as  crystals  by  the  action  of  caustic 
soda  on  haemoglobin  at  100°  C.  in  the  absence  of  oxygen  (Hoppe-Seyler). 
By  the  decomposition  of  haemoglobin  by  acids  (of  course  in  the  absence  of 
air)  we  obtain  hsemochromogen  contaminated  with  a  little  haematopor- 
phyrin. An  alkaline  hsemochromogen  solution  is  easily  obtained  by  the 
action  of  a  reducing  substance  (Stokes'  reduction  liquid)  on  an  alkaline 
haematin  solution. 

Haematin,  also  called  Oxyh^ematin,  is  sometimes  found  in  old  transu- 
dates. It  is  formed  by  the  action  of  the  gastric  or  pancreatic  juices  on  oxy- 
haemoglobin,  and  is  therefore  also  found  in  the  faeces  after  hemorrhage 
in  the  intestinal  canal,  and  also  after  a  meat  diet  and  food  rich  in  blood. 
It  is  stated  that  hsematin  may  occur  in  urine  after  poisoning  with  arseniu- 
retted  hydrogen.  As  shown  above,  the  haematin  is  formed  by  the  decom- 
position  of  oxyhaemoglobin.  or  at  least  of  haemoglobin,  in  the  presence  of 
oxygen. 

The  statements  in  regard  to  the  composition  of  haematin  are  rather 
contradictory,  which  seems  to  depend  upon  the  fact  that  different  haematins 
are  formed  under  various  conditions  (Kuster,  K.  Morner).  Cazeneuve 
and  Bretau  have  analyzed  hsematin  from  different  kinds  of  blood  (ox, 
horse,  sheep)  and  have  found  that  hsematin  from  a  certain  variety  of  blood 
has  the  same  composition,  while  that  from  a  different  species  of  animals 
has  a  different  composition.  According  to  Hoppe-Seyler  its  formula  is 
C34H35N4Fe05,  to  Nencki  and  Sieber,  which  corresponds  to  other  inves- 
tigators, the  formula  is  C32H32N4Fe04.  And  Kuster  finds  the  formula 
C31H3405N4Fe.  The  haematin  analyzed  by  K.  Morner  had  the  formula 
C35H36N4Fe03.  According  to  all  these  investigators  1  atom  of  iron  occurs 
with  every  4  atoms  of  nitrogen.  According  to  Cloetta,  and  also  Rosen- 
feld,2  haematin  has  the  formula  C30H34N3FeO3,  with  1  atom  of  iron  for 
every  3  atoms  of  nitrogen. 

1  Nord.  med.  Arkiv,  16. 

2  Hoppe-Seyler,  Med.  chem.  Untersuch.,  525;  Nencki  and  Sieber,  Arch.  f.  exp.  Path, 
u.  Pharm.,  18  and  20,  and  Ber.  d.  d.  chem.  Gesellsch.,  18;  Bialobrzeski,  Arch,  des 
scienc.  biol.  de  St.  Petersbourg,  5;  Kuster,  Beitriige  zur  Kenntniss  des  Haematins, 
Tubingen,  1896,  and  Ber.  d.  d.  chem.  Gesellsch.,  27  and  30,  and  Zeitschr.  f.  physiol, 
Chem.,  40;  K.  Morner,  Nord.  med.  Arkiv.  Festband.,  1897,  Nos.  1  and  26;  Cloetta, 
Arch.  f.  exp.  Path.  u.  Pharm.,  36;  Rosenfeld,  ibid.,  40;  Cazeneuve  and  Bretau,  Compt. 
rend.,  128. 


II. E  MATIN.  177 

Haematin  is  very  re  istant  towards  Kniling  concentrated  caustic  potash 
as  well  as  towards  boiling  hydrochloric  acid.  It  dissolves  in  concentrated 
sulphuric  acid  and  is  converted  into  luematoporphyrin  with  the  splitting 
off  of  iron.  On  heating  dry  hsematin  it  yields  abundant  pyrrol.  On 
reduction  with  tin  and  hydrochloric  acid  a  body  similar  to  urobilin  is 
formed.  As  oxidation  product  of  hsematin  in  glacial  acetic  acid  with 
potassium  bichromate  Kuster1  obtained  the  imide  of  the  tribasic  hacmatinic 
acid,  C8H9N04,  which  is  also  produced  on  the  oxidation  of  hsemotopor- 
phyrin  and  bilirubin. 

The  imide  of  the  tribasic  hacmatinic  acid  which  is  a  derivative  of  male'ic  acid 

CO 
has  probably  the  formula  C5H7(COOH)  <pq>NH,  is  readily  transformed  into 

the  anhydride  of  the  tribasic  hsematinic  acid,  C8H805,  having  the  probable  formula 
CH3.C.CO 

II        >0.     On   heating   the    imide  with  alcoholic   ammonia   to 
COOH.CH2.CH2.C.CO 

130°  C.  it  splits  off  carbon  dioxide  and  the  imide  of  the  bibasic  haematinic  acid, 
C7H0NO2,  is  obtained.  From  this  imide  on  saponification  with  baryta-water  we 
obtain  the  barium  salt  of  an  acid  whose   anhydride  is  methyl-ethyl  maleic-acid 

C2H5.C.CO 
anhydride,  II        >0. 

J  CH3.C.CO 

Haematin  is  amorphous,  dark  brown  or  bluish  black.  It  may  be  heated 
to  180°  C.  without  decomposition;  on  burning  it  leaves  a  residue  consisting 
of  iron  oxide  Ii  is  insoluble  in  water,  dilute  acids,  alcohol,  ether,  and 
chloroform,  but  it  dissolves  slightly  in  warm  glacial  acetic  acid.  Haematin 
dissolves  in  acidified  alcohol  or  ether.  It  easily  dissolves  in  alkalies,  even 
when  very  dilute.  The  alkaline  solutions  are  dichroitic;  in  thick  layers 
they  appear  red  by  transmitted  light  and  in  thin  layers  greenish.  The 
alkaline  solutions  are  precipitated  by  lime-  and  baryta-water,  as  also  by 
solutions  of  neutral  salts  of-  the  alkaline  earths.  The  acid  solutions  are 
always  brown . 

An  acid  haematin  solution  absorbs  the  red  part  of  the  spectrum  less  and 
the  violet  parts  more.  The  solution  shows  a  rather  sharply  defined  band 
between  C  and  D  whose  position  may  change  with  the  variety  of  acid  used 
as  a  solvent.  Between  D  and  F  a  second,  much  broader,  less  sharply 
defined  band  occurs  which  by  proper  dilution  of  the  liquid  is  converted  into 
two  bands.  The  one  between  b  and  F,  lying  near  F,  is  darker  and  broader, 
the  other,  between  D  and  E,  lying  near  E,  is  lighter  and  narrower.  Also 
by  proper  dilution  a  fourth  very  faint  band  is  observed  between  D  and  E, 
lying  near  D.  Haematin  may  thus  in  acid  solution  show  four  absorption- 
bands;  ordinarily  one  sees  distinctly  only  the  bands  between  C  and  D  and 
the  broad,  dark  band — or  the  two  bands — between  D  and  F.     In  alkaline 

1  W.  Kuster,  Ber.  d.  d.  chem.  Gesellsch.,  30,  32,  and  35;  Zeitschr.  f.  physiol.  Chem., 
28,  and  Annal.  d.  Chem.  u.  Pharm. ,  315. 


178  THE  BLOOD. 

solution  hsematin  shows  a  broad  absorption-band,  which  lies  in  greatest 
part  between  C  and  D,  but  reaches  a  little  over  the  line  D  towards  the 
right  in  the  space  between  D  and  E.  As  the  position  of  the  hsematin 
bands  in  the  spectrum  are  quite  variable,  the  exact  wave-lengths  corre- 
sponding thereto  cannot  be  given  exactly. 

Hsemin,  ELemin  Crystals,  or  Teichmann  's  Crystals.  Heemin  is  the 
hydrochloric-acid  ester  of  hsematin  and  is  the  starting-point  in  the  prepara- 
tion of  the  latter. 

The  statements  as  to  the  composition  of  hsemin  are  just  as  variable  as  those  for 
hsematin,  which  is  partly  due  to  the  fact,  as  shown  by  Nencki  and  Zaleski,1  that 
the  hsematin,  which  contains  two  hydroxyls  in  the  molecule,  may  form  ethers  with 
acids  and  alkyl  radicals,  which  also  yield  addition  products  with  indifferent 
compounds.  Thus  the  hsemin  prepared  according  to  Nencki  and  Sieber's 
method  contains  amyl  alcohol,  (C32H31N4Fe03Cl)4.C5H120.  Schalfejeff's  hsemin, 
having  the  formula  C:4H33N4Fe04Cl,  contains  an  acetyl  group  and  is  called  acet- 
haemin.  Morner's  hsemin,  C35H35N4Fe04Cl,  is  a  monethyl  ether  of  acethscmin 
and  has  the  formula  C3eH37N4Fe04Cl.  According  to  Kuster2  these  last-mentioned 
views  are  not  correct  and  the  question  as  to  Nencki  and  Zaleski  's  explanation 
of  these  contradictory  statements  requires  further  elucidation.  Kuster  3  finds 
that  the  hsemin  found  to  have  a  different  composition  by  various  investigators 
are  all  the  same  chemical  individual.  All  hsemins  have  the  same  empirical  formula, 
C34H3304X4FeCl,  hence  there  is  only  one  hsemin.  On  solution  of  the  hsemin  in 
alkali  an  intramolecular  change  takes  place.  By  the  action  of  boiling  aniline  upon 
haemin  HC1  and  H  are  expelled  and  aniline  is  taken  up  without  the  expulsion  of 
iron. 

Hsemin  crystals  form  in  large  masses  a  bluish-black  powder,  but  are  so 
small  that  they  can  only  be  seen  by  aid  of  the  microscope.    Thev  consist 

of  dark-brown  or  nearly  brownish-black  long,  rhombic,  or  spool-like  crystals, 
isolated  or  grouped  as  crosses,  rosettes,  or  stellar  forms.  Cubical  crystals 
may  also  occur,  according  to  Cloetta.  They  are  insoluble  in  water,  dilute 
acids  at  the  normal  temperature,  alcohol,  ether,  and  chloroform.  They.are 
slightly  soluble  in  glacial  acetic  acid  with  heat.  They  dissolve  in  acidi- 
fied alcohol,  as  also  in  dilute  caustic  or  carbonated  alkalies;  and  in  the 
last  case  they  form,  besides  alkali  chlorides,  soluble  hsematin  alkali,  from 
which  the  hsematin  may  be  precipitated  by  an  acid. 

The  principle  of  the  preparation  of  hsemin  crystals  in  large  quantities  is 
as  follows:  The  washed  sediment  from  the  blood-corpuscles  is  coagulated 
with  alcohol  or  by  boiling  after  dilution  with  water  and  the  careful  addition 
of  acid.  The  strongly  pressed  but  not  dry  mass  is  rubbed  with  90-95  per 
cent  alcohol  which  has  been  previously  treated  with  oxalic  acid  or  £-1  per 
cent  concentrated  sulphuric  acid,  and  this  is  allowed  to  stand  several  hours 
at  the  temperature  of  the  room.  The  filtrate  is  warmed  to  about  70°  C, 
treated  with  hydrochloric  acid  (for  each  liter  of  filtrate  add  10  c.  c.  25  per 


1  Zeitschr.  f.  physiol.  Chem.,  30.     See  also  foot-note  2,  page  176. 

2  Ber.  d.  d.  chem.  Gasellsch.,  .'}.">. 
8  Zeitschr.  f .  physiol.  Chem. ,  40. 


II.KM1X  AND  HjBMATOPORPHYRIN.  179 

cent  hydrochloric  acid  diluted  with  alcohol     Morner),  and    allowed   to 

stand  in  the  cold.  The  crystals,  which  separate  in  one  or  two  days,  are 
firgl  washed  with  alcohol  and  then  with  water.  For  particulars  as  in  the 
various  met  hods  we  refer  the  reader  to  the  cited  works  of  Nencki  and  Sieber, 

CLOfiTTA,    KtJSTER,   M6RNER,    RoSENFELD,    NENCKI    and    ZaLESKI    (SCHAL- 

vbjeff). 

Hannatin  is  obtained  on  dissolving  the  hsemin  crystals  in  very  dilute 
caustic  alkali  and  precipitating  with  an  acid. 

In  preparing  hsemin  crystals  in  small  quantities  proceed  in  the  following 
manner:  The  Mood  is  dried  after  the  addition  of  a  small  quantity  of  com- 
mon salt,  or  the  dried  blood  may  be  rubbed  with  a  trace  of  the  same. 
The  dry  powder  is  placed  on  a  microscope-slide,  moistened  with  glacial 
acetic  acid,  and  then  covered  with  the  cover-glass.  Add,  by  means  of  a 
glass  rod,  more  glacial  acetic  acid  by  applying  the  drop  at  the  edge  of  the 
cover-glass  until  the  space  between  the  slide  and  the  cover-glass  is  full. 
Now  warm  over  a  very  small  flame,  with  the  precaution  that  the  acetic  acid 
does  not  boil,  and  pass  with  the  powder  from  under  the  cover-glass.  If  no 
crystals  appear  after  the  first  warming  and  cooling,  warm  again,  and  if 
necessary  add  some  more  acetic  acid.  After  cooling,  if  the  experiment  has 
been  properly  performed,  a  number  of  dark-brown  or  nearly  black  hsemin 
crystals  of  varying  forms  will  be  seen. 

In  regard  to  the  preparation  of  iodohscmatin  and  the  use  of  the  same 
for  the  detection  of  blood  wre  must  refer  to  Strzyzowski's  communication.1 

By  the  action  of  acids  upon  hsemochromogen,  haematin,  or  hsemin  a 
new  iron-free  pigment,  which  was  first  closely  stttdied  by  Hoppe-Setleb 
and  called  hecmatoporphyrin,  is  produced.  According  to  the  method  of 
preparation  ha>matoporphyrins  having  different  solubilities  and  whose 
relationship  to  each  other  is  not  perfectly  clear,  are  produced,  but  all  show 
the  same  characteristic  absorption  spectrum.  The  best  studied  ha?mato- 
porphyrin  is  the  one  obtained  according  to  Xkxcki  and  Sieber 's  method, 
by  the  action  of  glacial  acetic  acid  saturated  with  hydrobromic  acid  upon 
hsemin  crystals,  best  at  the  temperature  of  the  body  (Nencki  and  Zalkski  2). 

Haematoporphyrin,  C1CH18N203,  or  Cadl^X/^  according  to  Zalkski3. 
This  pigment,  according  to  Mac  Mknx,4  occurs  as  a  physiological  pigment 
in  certain  animals.  It  occurs,  as  shown  by  Garrod  and  Saillet,  as  a  normal 
constituent,  although  only  as  traces,  of  human  urine.  It  occurs  in  greater 
quantities  in  human  urine  especially  after  the  use  of  sulphonal  (see  Chap- 
ter XV). 

The  formation  of  hsematoporphyrin  from  haematin  can  be  expressed 
by  the  following  equation  if  we  start  with  Nencki 's  formula  for  haematin: 
C32H32N4Fe04+2H204-2HBr=2C16H18N203-l-FeBr2+H2.  On  heating haemato- 

1  Therapeut.  Monatshefte,  1901  and  1902. 

1  Hoppe-Seyler,  Med.-chem.  Untersuch.,  528;  Nencki  ami  Sieber,  Monatshefte  f. 
Chem.,  9,  and  Arch.  f.  expt.  Path.  u.  Pharm.,  IS,  20,  and  24;  Nencki  and  Zaleski, 
Zeitschr.  f.  physiol.  Chem.,  30. 

3  Ibid.,  37,  54. 

4  Journ.  of  Physiol.,  7. 


ISO  THE  BLOOD. 

porphyrin  it  generates  an  odor  of  pyrrol.  On  oxidation  with  bichromate  and 
glacial  acetic  acid  it  yields  hsematinic  acid  (see  page  177).  A  pigment  closely 
alhed  to  the  urinary  pigment  urobilin  has  been  obtained  by  the  action  of 
reducing  substances  on  hsematoporphyrin  (Hoppe-Seyler,  Nencki  and 
Sieber,  Le  Nobel,  Mac  Munn).  On  the  administration  of  hsematopor- 
pyhrin  to  rabbits,  Nencki  and  Rotschy1  observed  that  a  part  was  reduced 
to  a  substance  similar  to  urobilin. 

Of  especial  interest  are  the  recent  investigations  of  Nencki,  Marchlewski 
and  Zaleski  2  upon  the  reduction  products  of  hsematoporphyrin  and  their  rela- 
tionship to  the  chlorophyll  derivatives.  By  the  action  of  glacial  acetic  acid  con- 
taining HI  and  iodophosphonium  upon  haemin  or  hsemochromogen  Nencki 
and  Zaleski  obtained  a  markedly  characteristic  pigment,  mesoporphyrin,  having 
the  formula  C16Hi,N202,  or,  according  to  Zaleski,3  C34H38N404,  and  which  stands 
in  a  certain  measure  between  hsematoporphyrin,  C18HlgN203,  and  the  chloro- 
phyll derivative  phylloporphyrin,  C16H18N20,  which  is  very  similar  to  hsemato- 
porphyrin. By  the  action  of  the  same  reducing  agent  upon  hsemin  or  hsemo- 
chromogen, but  under  other  conditions,  we  obtain  haemopyrrol,  C8H13N,  a  colorless 
oil,  which  in  the  air  gradually  changes  into  urobilin.  Hsemopyrrol  is  produced 
by  the  action  of  the  same  reducing  agents  upon  the  chlorophyll  derivative  phyllo- 
cyanin  (Nencki  and  Marchlewski),  which,  as  above  remarked,  shows  a  close 
relationship  between  the  blood  pigment  and  chlorophyll. 

Hsematoporphyrin  is,  according  to  Nencki  and  Sieber,  isomeric  with 
the  bile  pigment  bilirubin  and  like  this  latter  gives  a  play  of  colors — green, 
blue,  and  yellow — when  treated  with  fuming  nitric  acid.  The  hydrochloric- 
acid  combination  crystallizes  in  long  brownish-red  needles.  If  the  solution 
in  hydrochloric  acid  is  nearly  neutralized  with  caustic  soda  and  then  treated 
with  sodium  acetate,  the  pigment  separates  out  as  amorphous,  brown 
flakes  not  readily  soluble  in  amyl  alcohol,  ether,  and  chloroform,  but  readily 
soluble  in  ethyl  alcohol,  alkalies,  and  dilute  mineral  acids.  The  combina- 
tion with  sodium  crystallizes  as  small  tufts  of  brown  crystals.  The  acid 
alcoholic  solutions  have  a  beautiful  purple  color,  which  becomes  violet- 
blue  on  the  addition  of  large  quantities  of  acid.  The  alkaline  solution  has  a 
beautiful  red  color,  especially  when  not  too  much  alkali  is  present. 

An  alcoholic  solution  of  haematoporphyrin,  acidulated  with  hydrochloric 
or  sulphuric  acid,  shows  two  absorption-bands,  one  of  which  is  fainter  and 
narrower  and  lies  between  C  and  D,  near  D.  The  other  is  much  darker, 
sharper,  and  broader,  and  lies  in  the  middle  between  D  and  E.  An  absorp- 
tion extends  from  these  bands  towards  the  red,  terminating  with  a  dark 
edge,  which  may  be  considered  as  a  third  band  between  the  other  two. 

A  dilute  alkaline  solution  shows  four  bands,  namely,  a  band  between  C 
and  D;  a  second,  broader,  surrounding  D  and  with  its  broadest  part  between 

1  Hoppe-Seyler,  1.  c,  523;  Le  Nobel,  Pfliiger's  Arch.,  40;  Mac  Munn,  Proc.  Roy. 
Soc,  30,  and  Journ.  of  Physiol.,  10;  Nencki  and  Rotschy,  Monatshefte  f.  Chem.,  10. 

2  See  foot-note  2,  page  165. 

3  Zeitschr.  f.  physiol.  Chem.,  37. 


1LKMAT0ID1N.     DETECTION  OF  BLOOD.  181 

7)  and  E;  a  third  between  D  and  E,  nearly  at  E;  and  lastly,  a  fourth  broad 
and  dark  hand  between  b  and  F.  On  the  addition  of  an  alkaline  zinc- 
chloride  solution  the  spectrum  changes  more  or  less  rapidly,1  and  finally 
a  spectrum  is  obtained  with  only  two  bands,  one  of  which  surrounds  D 
and  the  other  lies  between  D  and  E.  If  an  acid  haematoporphyrin  solu- 
tion is  shaken  with  chloroform,  a  part  of  the  pigment  is  taken  up  by  the 
chloroform,  and  this  solution  often  shows  a  five-banded  spectrum  with 
two  bands  between  C  and  D.  The  position  of  the  haematoporphyrin  bands 
in  the  spectrum  differs  with  the  various  methods  of  preparation  and  other 
conditions,  so  that  they  do  not  correspond  to  the  same  wave-length. 

In  regard  to  the  preparation  of  haematoporphyrin,  see  Hoppe-Seyler- 
Thierfelder's  Handbuch,  7.  Aufl.,  and  the  works  cited  page  179. 

Haematinogen  is  a  ferruginous  pigment  so  named  by  Freund,2  which  he  ob- 
tained by  carefully  extracting  blood  with  alcohol  containing  hydrochloric  acid. 
It  is  closely  related  to  luematin,  but  is  not  sufficiently  characteristic  and  is  not 
considered  as  a  cleavage  product. 

Haematoidin,  thus  called  by  Virchow,  is  a  pigment  which  crystallizes 
in  orange-colored  rhombic  plates,  and  which  occurs  in  old  blood  extravasa- 
tions, and  whose  origin  from  the  blood-coloring  matters  seems  to  be  estab- 
lished (Langhans,  Cordua,  Quincke,  and  others  3).  A  solution  of  haema- 
toidin shows  no  absorption-bands,  but  only  a  strong  absorption  of  the 
violet  to  the  green  (Ewald  4).  According  to  most  observers,  haematoidin 
is  identical  with  the  bile-pigment  bilirubin.  It  is  not  identical  with  the 
crystallizable  lutein  from  the  corpora  lutea  of  the  ovaries  of  the  cow  (Pic- 
colo and  Lieben,5  Kuhne  and  Ewald). 

In  the  detection  of  the  above-described  blood-coloring  matters  the 
spectroscope  is  the  only  entirely  trustworthy  means  of  investigation.  If  it 
is  only  necessary  to  detect  blood  in  general  and  not  to  determine  definitely 
whether  the  coloring-matter  is  haemoglobin,  methaemoglobin,  or  haematin, 
then  the  preparation  of  haemin  cr}rstals  is  an  absolutely  positive  proof.  The 
reader  is  referred  to  more  extended  text-books  for  exacter  methods  for  the 
detection  of  blood  in  chemico-legal  cases,  and  it  is  perhaps  sufficient  to  give 
here  the  chief  points  of  the  investigation. 

If  spots  on  clothes,  linen,  wood,  etc.,  are  to  be  tested  for  the  presence  l 
of  blood,  it  is  best,  when  possible,  to  scratch  or  shave  off  as  much  as  pos- 
sible, rub  with  common  salt,  and  from  this  prepare  the  haemin  crystals. 
On  obtaining  positive  results  the  presence  of  blood  is  not  to  be  doubted. 
When  sufficient  material  is  not  obtained  by  the  above  means,  soak  the  spot 
with  a  few  drops  of  water  in  a  watch-crystal.     If  a  colored  solution  is 


1  See  Hammarsten,  Skand.  Arch.  f.  Physiol.,  3,  and  Garrod  Journ.  of  Physiol.,  13. 

2  Wien.  klin.  Wochenschr.,  1903. 

3  A  comprehensive  review  of  the  literature  pertaining  to  haematoidin  may  be  found 
in  Stadelmann:  Der  Icterus,  etc.     Stuttgart,  1S91.     Pages  3  and  45. 

4  Zeitschr.  f.  Biologie,  22,  475. 

6  Cit.  from  Gorup-Besanez :  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,  1878. 


182  THE  BLOOD. 

thus  obtained,  then  remove  the  fibres,  wood-shavings,  and  the  like  as  far  as 
possible,  and  allow  the  solution  to  dry  in  the  watch-glass.  The  dried 
residue  may  be  partly  used  for  the  spectroscope  test  directly,  and  part 
may  be  employed  in  the  preparation  of  the  hsemin  crystals.  It  may  also 
be  used  to  detect  hsemochromogen  in  alkaline  solution  after  previous  treat- 
ment with  alkali  and  the  addition  of  reducing  substances. 

If  a  colorless  solution  is  obtained  after  soaking  with  water,  or  the  spots 
are  on  rusty  iron,  then  digest  with  a  little  dilute  alkali  (5  p.  m.).  In  the 
presence  of"  blood  the  solution  gives,  after  neutralization  with  hydrochloric 
acid  and  drying,  a  residue  which  may  give  the  hsemin  crystals  with  glacial 
acetic  acid.  Another  part  of  the  alkaline  solution  shows,  after  the  addi- 
tion of  Stokes'  reduction  fluid,  the  absorption-bands  of  hsemochromogen 
in  alkaline  solution. 

The  methods  proposed  for  the  quantitative  estimation  of  the  blood- 
coloring  matters  are  partly  chemical  and  partly  physical. 

Among  the  chemical  methods  to  be  mentioned  is  the  ashing  of  the  blood 
and  the  determination  of  the  amount  of  iron  contained  therein,  from  which  the 
amount  of  haemoglobin  may  be  calculated.  Jolles  *  has  recently  suggested  a 
clinical  method  based  on  the  incineration  of  the  blood  and  determination  of  the 
iron  in  the  ash. 

The  physical  methods  consist  either  in  a  colorimetric  or  a  spectroscopic 
investigation. 

The  principle  of  Hoppe-Seyler's  colorimetric  method  is  that  a  measured 
quantity  of  blood  is  diluted  with  an  exactly  measured  quantity  of  water 
until  the  diluted  blood  solution  has  the  same  color  as  a  pure  oxyhsemo- 
globin  solution  of  a  known  strength.  The  amount  of  coloring  matter 
present  in  the  undiluted  blood  may  be  easily  calculated  from  the  degree  of 
dilution.  In  the  colorimetric  testing  we  use  a  glass  vessel  with  parallel 
sides  containing  a  layer  of  liquid  1  cm.  thick  (  Hoppe-Seyler's  hsematinom- 
eter).  The  use  of  Hoppe-Seyler's  colorimetric  double  pipette  is  more 
advantageous.  Other  good  apparatus  have  been  constructed  by  Giacosa 
and  Zangermeister.2  Instead  of  an  oxyhsemoglobin  solution  we  now  gen- 
erally use  a  carbon-monoxide  hsemoglobin  solution  as  comparison  liquid 
because  it  may  be  kept  for  a  long  time.  The  blood  solution  in  this  case 
is  saturated  with  carbon  monoxide.3 

The  quantitative  estimation  of  the  blood-coloring  matters  by  means  of 
the  spectroscope  may  be  done  in  different  ways,  but  at  the  present  time 
the  spectrophotometry  method  is  chiefly  used,  and  this  seems  to  be  the  most 
reliable.  This  method  is  based  on  the  fact  that  the  extinction  coefficient 
of  a  colored  liquid  for  a  certain  region  of  the  spectrum  is  directly  propor- 
tional to  the  concentration,  so  that  C  :E  =  C1  :Elf  when  C  and  Ct  repre- 
sent the  different  concentrations  and  E  and  Et  the  corresponding  coefficients 

1  Pfliiger's  Arch.,  65,  and  Monatshefte  f.  Chem.,  17. 

2  F.  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  16;  G.  Hoppe-Seyler,  ibid.,  21; 
Winternitz,  ibid.;  Giacosa,  Maly's  Jahresber.,  26;  Zangermeister,  Zeitschr.  f.  Biolo- 
gic, 33. 

3  See  Hal dane,  Journ.  of  Physiol.,  26. 


SPECTROPHOTOMETRY.  ls:; 

c    c 

of  extinction.     From  the  equation  y  =  p\  it  follows  that  for  one  and  the 

same  pigment  this  relation,  which  is  called  the  absorption  ratio,  must  be 
constant.  It"  the  absorption  ratio  is  represented  by  A,  the  determined 
extinction  coefficient  by  E,  and  the  concentration  (the  amount  of  coloring 

matter  in  grams  in  1  c.  c.)  by  C,  then  C  =  A  .  E. 

Different  apparatus  have  been  constructed  (Vierordt  and  Hufner1) 
for  the  determination  of  the  extinction  coefficient  which  is  equal  to  the 

negative  logarithm  of  those  rays  of  light  which  remain  after  the  p 

the  light  through  a  layer  1  cm.  thick  of  an  absorbing  liquid.     In  regard  to 

this  apparatus  the  reader  is  referred  to  other  text-books. 

As  control  the  extinction  coefficients  are  determined  in  two  different  regions 
of  the  spectrum.  HuiNBB  has  selected  (a)  the  region  between  the  two  absorption- 
bands  of  oxyhemoglobin,  especially  between  the  wave-lengths  554  /jl/x  and  565  llll, 
and  (/))  the  region  between  the  two  bands,  especially  the  interval  between  the  wave 
lengths  531.5  ufl  and  542.5  111.1.  The  constants  or  the  absorption  ratio  for  these 
two  regions  of  the  spectrum  are  designated  by  Hufner  by  A  and  A'.  Before 
the  determination  the  blood  must  be  diluted  with  water,  and  if  the  proportion 
of  dilution  of  the  blood  be  represented  by  V,  then  the  concentration  or  the  amount 
of  coloring  matter  in  100  parts  of  the  undiluted  blood  is 

C-100.V .A.E  and 
C  =  100.  V.A'.E'. 

■  The  absorption  ratio  or  the  constants  in  the  two  above-mentioned  regions 
of  the  spectrum  have  been  determined  for  oxyhemoglobin,  haemoglobin,  carbon 
monoxide,  andmethemoglobin,  as  follows: 

Oxyhemoglobin .4.  =0.002070  and  A'a  =0.001312 

Hemoglobin Ar  =0.001354  and  A'r  =0.001778 

Carbon-monoxide  hemoglobin.  .  .  .4,  =0.001383  and  A'c  =0.001263 

Methannoglobin Am  =0.002077  and  .4 'm  =0.00175  L 

The  quantity  of  each  coloring  matter  majr  be  determined  in  a  mixture  of 
two  blood-coloring  matters  by  this  method;  this  is  of  special  import  ante  in 
the  determination  of  the  quantity  of  oxyhemoglobin  and  hemoglobin  present 
in  blood  at  the  same  time. 

In  order  to  facilitate  these  determinations  Hufner2  has  worked  out  tables 
which  give  the  relation  between  the  two  pigments  existing  in  a  solution  contain- 
ing oxyhemoglobin  and  another  pigment  (hemoglobin,  nictlucmoglobin,  or  carbon- 
monoxide  hemoglobin),  and  thus  allowing  of  the  calculation  of  the  absolute  quan- 
tity of  each  pigment. 

Among  the  many  apparatus  constructed  for  clinical  purposes  for  the 
quantitative  estimation  of  haemoglobin,  Fleischl's  hoemometer,  which  has 
undergone  numerous  modifications,  and  Henocque's  hccinatoscope  are  to  be 
specially  mentioned.  In  regard  to  these  apparatus,  see  v.  Jaksch,  Klinische 
Diagnostik  innerer  Krankheiten,  4  Auflage,  1897.  and  Jaquet,  Corresp. 
Blatt.  f.  Schweiz.  Aerzte,  1897;  Gartner,  Munchener  Med.  "VVochen- 
schr.,  1901. 

1  See  Vierordt,  Die  Anwendung  des  Spektralapparates  zu  Photometrie,  etc.  (Tubin- 
gen, 1873),  and  Hufner,  Du  Bois-Reymond's  Arch.,  1894,  and  Zeitschr.  f.  physiol.  Chem., 
3;  v.  Noorden,  ibid.,  4;  Otto,  Pfluper's  Arch.,  31  and  36. 

'Arch.  f.  (Anat.  u.)  Physiol.,  1900. 


184  THE  BLOOD. 

Many  other  pigments  are  found  besides  the  often-occurring  haemoglobin 
in  the  blood  of  invertebrates.  In  a  few  Arachnidae,  Crustacea,  Gasteropodae, 
and  Cephalopoda^  a  body  analogous  to  haemoglobin,  containing  copper,  hcemo- 
cyanin,  has  been  found  by  Fredericq.  By  the  taking  up  of  loosely  bound  oxygen 
this  body  is  converted  into  blue  oxyhcemocyanin,  and  by  the  escape  of  the  oxygen 
becomes  colorless  again.  According  to  Henze  1  grm.  haemoeyanin  combines 
with  about  0.4  c.  c.  oxygen.  It  is  crystalline  and  has  the  following  composition: 
C  53.66;  H  7.33;  N  16.09;  S  0.86;  Cu0.38;  0  21.67  per  cent.  A  coloring  matter 
called  chlorocruorin  by  Lankester  is  found  in  certain  Chaetopodae.  Hoemerythrin, 
so  called  by  Krukenberg  but  first  observed  by  Schwalbe,  is  a  red  coloring 
matter  from  certain  Gephyrea.  Besides  haemoeyanin  we  find  in  the  blood  of 
certain  Crustacea  the  red  coloring  matter  tetronerythrin  (Halliburton),  which 
is  also  widely  spread  in  the  animal  kingdom.  Echinochrom,  so  named  by  Mac- 
Munn,1  is  a  brown  coloring  matter  occurring  in  the  perivisceral  fluid  of  a  variety 
of  echinoderms. 

The  quantitative  constitution  of  the  red  blood-corpuscles.  The  amount 
of  water  varies  in  different  varieties  of  blood  between  570-644  p.  m.,  with 
a  corresponding  amount,  430-356  p.  m.,  of  solids.  The  chief  mass,  about 
iV-tV'  of  the  dried  substance  consists  of  haemoglobin  (in  human  and  mam- 
malian blood). 

According  to  the  analyses  of  Hoppe-Seyler  2  and  his  pupils,  the  red. 

corpuscles  contain  in  1000  parts  of  the  dried  substance: 

• 

Haemoglobin.  Proteid.  Lecithin.  Cholesterin. 

Human    blood 868-944  122-51  7.2-3.5            2.  5 

—TJog"^          "    '865                  126  5.9                 3.6 

Goose's       "    627                  364  4.6                 4.8 

Snake's       "     467  525 

Abderhalden  found  the  following  composition  for  the  blood-corpuscles 
from  the  domestic  animals  investigated  by  him:  Water,  591.9-644.3 
p.  m.;  solids,  408.1-355.7  p.  m.;  haemoglobin,  303.3-331.9  p.  m.;  proteid, 
5.32  (dog)-78.5  p.  m.  (sheep);  cholesterin,  0.388  (horse)-3.593  p.  m. 
(sheep);  and  lecithin,  2.296  (dog)-4.855  p.  m. 

Of  special  interest  is  the  varying  proportion  of  the  haemoglobin  to  the 
proteid  in  the  nucleated  and  in  the  non-nucleated  blood-corpuscles.  These 
last  are  much  richer  in  haemoglobin  and  poorer  in  proteid  than  the  others. 

The  amount  of  mineral  bodies  in  various  species  of  animals  is  different. 
According  to  Bunge  and  Abderhalden  the  red  corpuscles  from  the  pig, 
horse,  and  rabbit  contain  no  soda,  while  those  from  man,  the  ox,  sheep,  goat 
dog,  and  cat  are  relatively  rich  in  soda.  In  the  five  last-mentioned  species 
the  amount  of  soda  was  2.135-2.856  p.  m.  The  quantity  of  potash  was 
0.257  (dog)-0.744  p.  m.  (sheep).     In  the  horse,  pig,  and  rabbit  the  quantity 

1  Fredericq,  Extrait  des  Bulletins  de  l'Acad.  Roy.  de  Belgique  (2),  46,  1878;  Lan- 
kester, Journ.  of  Anat.  and  Physiol.,  2  and  4;  Henze,  Zeitschr.  f.  physiol.  Chem.,  33; 
Krukenberg,  see  Vergl.  Physiol.  Studien,  Reihe  1,  Abth.  3.  Heidelberg,  1880;  Halli- 
burton, Journal  of  Physiol.,  6;  MacMunn,  Quart.  Journ.  Microsc.  Science,  1885. 

*Med.-chem.  Untersuch.,  390  and  393. 


WHITE  CORPUSCLES.  185 

of  potash  was  3.326  (horse)-5.229  p.  m.  (rabbit).  Human  blood-corpuscles 
contain,  according  to  Wanach,  about  five  times  as  much  potash  as  soda, 
OD  an  average  3.99  p.  m.  potash  and  0.75  p.  in.  soda.  The  nucleated 
erythrocytes  of  the  frog,  toad,  and  turtle  contain,  according  to  Bottazzi 
and  Cappelli,1  also  considerably  more  potassium  than  sodium.  Lime  is 
claimed  to  be  absent  in  the  blood-corpuscles,  and  magnesia  occurs  only 
in  small  amounts:  0.016  (sheep)-0.150  p.  m.  (pig).  The  blood-corpuscles 
of  all  animals  investigated  contain  chlorine,  0.460-1.949  p.  m.  (both  in  horse), 
generally  1  to  2  p.  m.,  and  also  phosphoric  acid.  The  amount  of  inorganic 
phosphoric  acid  shows  great  variation:  0.275  (sheep)-1.916  p.  m.  (horse). 
All  above  figures  are  calculated  on  the  fresh,  moist  blood-corpuscles. 

By  quantitative  determinations  of  the  swelling  and  shrinking  of  the  cells 
under  the  influence  of  NaCl  solutions  of  various  concentration  or  of  serum  of 
various  dilutions,  Hamburger  has  attempted  to  determine  for  the  erythrocytes, 
as  well  as  the  leucocytes,  the  percentage  relationship  between  the  two  chief  con- 
stituents of  the  cells  (the  frame  and  the  intracellular  fluid).  He  found  that  the 
volume  of  the  frame  substance  for  both  varieties  of  blood-corpuscles  of  the  horse 
rjual  to  53-56.1  per  cent.  The  volume  for  the  red  blood-corpuscles  was 
for  the  rabbit  48.7-51 ;  hen,  52.4-57.7,  and  for  the  frog,  72-76.4  per  cent.  Koeppe 
has  raised  objections  to  these  determinations.2 

The  White  Blood-Corpuscles  and  the  Blood-Plates. 

The  White  Blood-corpuscles,  also  called  TKTTmovTps  or  Lymphoid 
fV11g|  ^hicb  ocr.nr  in  t.hf»  blood  in  varying  shapes^  and  sizes,  form  in  a  state 
of  rest  spherical  lumps  of  a  sticky,  highly  refractive,  non-membranous 
protoplasm,  capable  of  motion,  and  which  show  1-4  nuclei  on  the  addi- 
tion of  water  or  acetic  acid.  In  human  and  mammalian  blood  they  are 
larger  than  the  red  blood-corpuscles.  They  have  also  a  lower  specific 
gravity  than  the  red  corpuscles,  mflve  in  the  circulating  blood  nearer  to  the 
walls  of  the  blood-vessels,  and  have  also  a  slower  motion. 

The  number  of  white  blood-corpuscles  varies  not  only  in  the  different 
blood-vessels,  but  also  under  different  physiological  conditions.  As  an 
average  there  is  only  1  white  corpuscle  for  350-500  red  corpuscles.  Accord- 
ing to  the  investigations  of  Alex.  Schmidt  3  and  his  pupils,  the  leucocytes 
are  destroyed  in  great  part  on  the  discharge  of  the  blood  before  and  during 
coagulation,  so  that  discharged  blood  is  much  poorer  in  leucocytes  than 
the  circulating  blood.  The  correctness  of  this  statement  has  been  denied 
by  other  investigators . 

From  a  histological  standpoint  we  generally  discriminate  between  the 

1  Bunge,  Zeitschr.  f.  Biologie,  12,  and  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  23 
and  25;  Wanach,  Maly's  Jahresber.,  IS,  88;  Bbttazzi  and  Cappelli,  Arch.  Ital.  de  Biolo- 
gie, 32. 

2  Hamburger,  Arch.  f.  (Anat.  u.)  Physiol.,  1898;   Koeppe,  ibid.,  1899  and  1900. 
1  Pfliiger's  Arch.,  11. 


186  .  THE  BLOOD. 

different  kinds  of  colorless  blood-corpuscles;  chemically  considered,  how- 
ever, there  is  no  known  essential  difference  between  them.  With  regard  to 
their  importance  in  the  coagulation  of  fibrin  Alex.  Schmidt  and  his  pupils 
distinguish  between  the  leucocytes  which  are  destroyed  by  the  coagulation 
and  those  which  are  not.  The  last  mentioned  give  with  alkalies  or  common- 
salt  solutions  a  slimy  mass;  the  first  do  not  show  such  behavior. 

The  protoplasm  of  the  leucocytes  has  during  life  amoeboid  movements 
which  partly  make  possible  the  wandering  of  the  cells  and  partly  the  taking 
up  of  smaller  grains  or  foreign  bodies  within  the  same.  On  these  grounds 
the  occurrence  of  myosin  in  them  has  been  admitted  even  without  any 
special  proof  thereof.  Alex.  Schmidt  claims  to  have  found  serglobulin  in 
equine-blood  leucocytes  which  have  been  washed  with  ice-cold  water.  There 
are  also  certain  leucocytes,  as  above  stated,  which  yield  a  slimy  mass  when 
treated  with  alkalies  or  NaCl  solutions,  which  seems  to  be  identical  with  the 
so-called  hyaline  substance  of  Rovida  found  in  the  pus-cells.  On  digesting 
the  leucocytes  with  water,  a  solution  of  a  protein  body  is  obtained  which  can 
be  precipitated  by  acetic  acid,  and  forms  the  chief  mass  of  the  leucocytes. 
This  substance,  which  is  undoubtedly  concerned  in  the  coagulation  of  the 
blood,  has  been  described  under  different  names  (see  Chapter  V,  page  116), 
and  consists,  chiefly  at  least,  of  nucleoproteid.  The  ordinary  view  that 
this  is  nucleohiston  does  not  seem  to  be  correct,  according  to  the  recent 
investigations  of  Bang,1  and  further  proof  is  necessary. 

Glycogen,  as  above  stated,  is  found  in  the  leucocytes.  The  glycogen 
found  by  Huppert,  CzeAny,  Dastre,2  and  others  in  blood  and  lymph 
probably  originated  from  the  leucocytes.  The  constituents  of  the  leuco- 
cytes are  the  same  as  the  constituents  of  the  cell  as  described  in  Chapter  V. 

The  blood-plates  (Bizzozero's),  hsematoblasts  (Hayem),  whose  nature, 
preformed  occurrence,  and  physiological  importance  have  been  much  ques- 
tioned, are  pale,  colorless,  gummy  disks,  round  or  more  oval  in  shape  and 
generally  with  a  diameter  two  or  three  times  smaller  than  the  red  blood- 
corpuscles.  The  blood-plates  separate  into  two  substances  by  the  action  of 
different  reagents,  namely,  one  which  is  homogeneous  and  non-refractive, 
while  the  other  is  highly  refractive  and  granular.  Blood-plates  readily 
stick  together  and  attach  themselves  to  foreign  bodies. 

According  to  the  researches  of  Kossel  and  of  Lilienfeld  3  the  blood- 
plates  consist  of  a  chemical  combination  between  proteid  and  nuclein, 
and  hence  they  are  also  called  nuclein-plates  by  Lilienfeld,  and  are  con- 

1  I.  Bang,  Studier  over  Nukleoproteider.     Kristiania,  1902. 

2  Huppert,  Centralbl.  f.  Physiol.,  6,  394;  Czerny,  Arch.  f.  exp.  Path.  u.  Pharm.,  31; 
Dastre,  Compt.  rend.,  120,  and  Arch,  de  Physiol.  (5),  7. 

3  In  regard  to  the  literature  of  the  blood-plates,  see  Lilienfeld,  Du  Bois-Reymond's 
Archiv,  1892,  and  "Leukocyten  und  Blutgerinnung, "  Verhandl.  d.  physiol.  Gesellsch. 
zu  Berlin,  1892;  and  also  Mosen,  Du  Bois-Reymond's  Arch.,  1893,  and  Maly's  Jahres- 
ber.,  30  and  31. 


SPECIFIC  GRAVITY  AND  REACTION.  187 

sidered  as  derivatives  of  the  cell  nucleus.  It  seems  certain  that  the  blood- 
plates  stand  in  a  certain  relationship  to  the  coagulation  of  blood,  and 
according  to  Lilienfeld  the  fibrin  coagulation  is  indeed  a  function  of  the 
cell  nucleus.     The  views  on  this  subject  are  unfortunately  very  divergent. 

III.  THE  BLOOD  AS  A  MIXTURE  OF  PLASMA  AND  BLOOD-CORPUSCLES. 


The  blood  in  itself  is  a  thick,  sticky,  light  or  dark  red  liquid, -opaque 
even  in  thin  layers  r  having  a  salty  taste  and  a  faint  odor  differing  in  differ- 
ent kinds  of  animals.  On  the  addition  of  sulphuric  acid  to  the  blood  the 
odor  is  more  pronounced.  In  adult  human  beings  the  specific  gravity 
ranges  between  1.045  and  1075-  It  has  an  average  of  1.058  for  grown 
men  and  a  little  less  for  women.  Lloyd  Jones  found  that  the  specific 
gravity  is  highest  at  birth  and  lowest  in  children  when  about  two  years  old 
and  in  pregnant  women.  The  determinations  of  Lloyd  Jones,  Hammer- 
schlag,1  and  others  show  that  the  variation  of  the  specific  gravity,  depend- 
ent upo  and  sex,  corresponds  to  the  variation  in  the  quantity  of 
haemoglobin. 

The  determination  of  the  specific  gravity  is  most  accurately  done  by 
means  of  the  pyknometer.  For  clinical  purposes,  where  only  small  amounts 
are  available,  it  is  best  to  proceed  with  the  method  as  suggested  by  Mam- 
MF.RscHLAq.  Prepare  a  mixture  of  chloroform  and  benzene  of  about  1.050 
sp.  gr.  and  add  a  drop  of  the  blood  to  this  mixture.  If  the  drop  rises  to 
the  surface  then  add  benzene,  and  if  it  sinks  add  chloroform.  Continue 
this  until  the  drop  of  blood  suspends  itself  midway  and  then  determine 
the  specific  gravity  of  the  mixture  by  means  of  an  areometer.  This  method 
is  not  strictly  accurate  and  must  be  performed  quickly^  In  regard  to  the 
necessary  details  refer  to  Zuntz  and  A.  Levy.1 

The  reaction  of  the  blood  is  alkaline  towards  litmus.  The  quantity  of 
alkali,  calculated  as  Na^CO,,,  in  fresh,  non-defibrinated  blood  from  the 
dog,  horse,  and  man  is,  according  to  Loewy,  4.93,  4.43,  and  5.95  p.  m. 
respectively.  According  to  Strauss,  the  average  for  normal  human 
blood  may  be  calculated  as  about  4.43  p.  m.  Na2C03.  '  Below  3.3  p.  m. 
and  above  5.3  p.  m.  are,  according  to  him,  to  be  considered  as  pathological, 
v.  Jaksch  found  the  quantity  of  alkali  in  man  to  vary  between  3.38  and 
3.90  p.  m.  and  Brandenburg  found  3  p.  m.  NaOH  (  =  3.98  p.  m.  NajjCOj). 
The  alkaline  reaction  diminishes  outside  of  the  body,  and  indeed  the  more 
quickly,  the  greater  the  original  alkalinity  of  the  blood.  This  depends  on 
the  formation  of  acid  in  the  blood,  in  which  the  red  blood-corpuscles  seem 
to  take  part  in  some  way  or  another.  After  excessive  muscular  activity  the 
alkalinity   is   diminished   (Peiper,   Cohnstein),   and  it   is   also   decreased 

1  Lloyd  Jones,  Journ.  of  Physiol.,  8;  Ilammerschlag,  Wien.  klin.  Wochenschrift, 
1890,  and  Zeitschr.  f.  klin.  Med.,  20. 

:Zu:itz,  Pfliiger's  Arch.,  66;    Levy,  Proceed.  Roy.  Soc,  81. 


188  THE  BLOOD. 

after  the  continuous  use  of  acids  (Lassar,  Freudberg  *).  Numerous  inves- 
tigations have  been  made  in  regard  to  the  alkalinity  of  the  blood  in  disease, 
but  as  there  is  at  present  no  trustworthy  method  for  estimating  the  alkalinity 
of  the  blood,  these  investigations,  as  also  the  statements  in  regard  to  the 
physiological  alkalinity,  require  further  substantiation.2  Attention  must 
also  be  called  to  what  was  stated  (page  160)  in  regard  to  the  determination 
of  the  alkalinity  of  blood-serum — that  determinations  are  made  only  of  the 
titratable  alkali  and  not  of  the  true  alkalinity  caused  by  hydroxyl  ions. 

The  alkali  of  the  blood  exists  in  part  as  alkaline  salts,  carbonate  and 
phosphate,  and  part  in  combination  with  proteid  or  hsemoglobin.  The  first 
are  often  spoken  of  as  readily  diffusible  alkalies,  while  the  others  are  not,  or 
are  only  diffusible  with  difficulty  (see  page  157).  The  readily  as  well  as 
the  difficultly  diffusible  alkali  is  divided  between  the  blood-corpuscles  and 
plasma,  and  the  blood-corpuscles  seem  to  be  richer  in  difficultly  diffusible 
alkali  than  the  plasma  or  serum.  This  division  may  be  changed  by  the 
influence  of  even  very  small  amounts  of  acid,  also  carbon  dioxide,  and  also, 
as  shown  by  Zuntz,  Loewy  and  Zuntz,  Hamburger,  Limbeck  and  Gurber,3 
by  the  influence  of  the  respiratory  pynhanp-p  of  gas.  The,  Mood-nnrpuscles 
give  up  a  part  of  the  alkali  united  with  proteid  to  the  serum  by  the  action 
of  carbon  dioxide,  hence  the  serum  becomes  more  alkaline.  The  equi- 
librium of  the  osmotic  tension  in  the  blood-corpuscles  and  in  the  serum 
is  hereby  destroyed;  the  blood-corpuscles  swell  up  because  they  take  up 
water  from  the  serum  and  this  then  becomes  more  concentrated  and  richer 
in  alkali,  proteid,  and  sugar.  Under  the  influence  of  oxygen  the  corpuscles 
take  their  original  form  again  and  the  above  changes  are  restored.  The 
blood-corpuscles  for  this  reason  are  less  biconcave  in  their  small  diameter  in 
venous  than  in  arterial  blood  (Hamburger). 

The  color  of  the  blood  is  red — light  scarlet-red  in  the  arteries  and  dark 
bluish-red  in  the  veins.  Blood  free  from  oxygen  is  dichroitic,  dark  red 
by   reflected  light,   and  green  by   transmitted  light.     The  blood-coloring 


1  Loewy,  Pfluger's  Arch.,  58,  which  also  contains  the  references  to  the  literature; 
H.  Strauss,  Zeitschr.  f.  klin.  Med.,  30;  v.  Jaksch,  ibid.,  13;  Peiper,  Virchow's  Arch., 
110;  Cohnstein,  ibid.,  130,  which  also  cites  the  works  of  Minkowski,  Zuntz,  and  Gep- 
pert;  Freudberg,  ibid.,  125.  See  also  Weiss,  Zeitschr.  f.  physiol.  Chem.,  38;  Branden- 
burg, Zeitschr.  f.  klin.  Med.,  45. 

2  In  regard  to  the  methods  for  the  estimation  of  the  alkalinity  see,  besides  the  above- 
mentioned  authors,  v.  Jaksch,  Klin.  Diagnostik;  v.  Limbeck,  Wien.  med.  Blatter,  18; 
Wright,  The  Lancet,  1897;  Biernacki,  Beitrage  zur  Pncumatologie,  etc.,  Zeitschr.  f. 
klin.  Med.,  31  and  32;  Hamburger,  Eine  Methode  zur  Trennung,  etc.,  Du  Bois-Rey- 
mond's  Arch.,  1898.     See  also  Maly's  Jahresber.,  29,  30,  and  31. 

3  Zuntz  in  Hermann's  Handbuch  der  Physiol.,  4,  Abth.  2;  Loewy  and  Zuntz, 
Pfiiiger's  Arch.,  58;  Hamburger,  Du  Bois-Reymond 's  Arch.,  1894  and  1898,  and 
Zeitschr.  f.  Biologie,  28  and  35;  v.  Limbeck,  Arch.  f.  exp.  Path.  u.  Pharm.,  35;  Gurber, 
Sitzungsber.  d.  phys.  med.  Gesellsch.  zu  Wiirzburg,  1895. 


COAGULATION  OF   THE  BLOOD.  1*9 

matters  occur  in  the  blood-corpuscles.     For  this  reason  blood  jg  gpaque  ia_ 

thin  layers  and  acts  as"  a  "deck-Jarbc."  If  tin-  haemoglobin  is  removed 
from  the  stroma  and  dissolved  by  the  blood  liquid  by  any  of  the  above- 
mentioned  means  (see  page  1fi2),  t.hf>  blood  hen m us  transparent  and  acts 
then  like  a  "  lake  color,"  Less  light  is  now  reflected  from  its  interior,  and 
thus  laky  blood  Is  therefore  darker  in  thicker  layers.  On  the  addition  of 
salt  solutions  to  the  blood-corpuscles  they  shrink  and  more  light  Ls  reflected 
and  the  color  appears  lighter.  .A.  great  abundance  of  red  corpuscles  makes 
the  blood  darker,  while  by  diluting  with  serum  or  by  a  greater  abundance 
of  white  corpuscles  the  blood  becomes  lighter  in  appearance.  The  different 
colors  of  arterial  and  of  venous  blood  depend  on  the  varying  quantity  of 
gas  contained  in  these  two  varieties  of  blood,  or,  better,  on  the  different 
amounts  of  oxyhemoglobin  and  haemoglobin  they  contain. 

The  most  striking  property  of  blood  consists  in  its  coagulating  within  a 
shorter  or  longer  time,  but  as  a  rule  very  shortly  after  leaving  the  vein. 
Different  kinds  of  blood  coagulate  with  varying  rapidity;  in  human  blood 
the  first  marked  sign  of  coagulation  is  seen  in  two  to  three  minutes,  and  within 
seven  to  eight  minutes  the  blood  is  thoroughly  converted  into  a  gelatinous 
mass^  If  the  blood  is  allowed  to  coagulate  slowly,  the  red  corpuscles  have 
time  to  settle  more  or  less  before  the  coagulation,  and  the  blood-clot  then  __ 
shows  an  upper  ycllowish-gra\T  or  reddish-gray  layer  consisting  of  fibrin  en- 
closing chiefly  colorless  corpuscles.  This  layer  has  been  called  crusta  inflam- 
matona  or  vhloaistica.  bgcause  it  has  been  especially  observed  in  inflan> 
matory  processes  and  is  considered  one  of  the  characteristics  of  them.  This 
frusta,  or  "huffy  coat,"  is  not  characteristic  of  any  special  disease  and  it 
occurs  chiefly  when  the  blood  coagulates  slowly  or  when  the  blood-corpuscles 
settle  more  quickly  than  usual.  A  buffy  coat  is  often  observed  in  the  slow- 
coagulating  equine  blood.  The  blood  from  the  capillaries  is  not  supposed 
to  have  the  power  of  coagulating. 

Coagulation  is  retarded  by  cooling,  by  diminishing  the  oxygen  and 
increasing  the  amount  of  carbon  dioxide,  which  is  the  reason  that  venous 
blood  and  to  a  much  higher  degree  blood  after  asphyxiation  coagulates 
more  slowly  than  arterial  blood.  The  coagulation  may  be  retarded  or  pre- 
vented by  the  addition  of  acids,  alkalies,  or  ammonia,  even  in  small  quanti- 
ties^ by  concentrated  solutions  of  neutral  alkali,  salts  and  alkaline  earths7 
alkali  oxalates  and  fluorides;  also  by  egg-albumin,  solutions  of  sugar  or 
gum,  glycerine,  or  much  water;  also  by  receiving  the  blood  in  oil.  Coagula* 
tion  may  be  prevented  by  the  injection  of  a  proteose  solution  or  of  aji 
"infusion  of  the  leech  into  the  circulating  blood,  but  this  infusion  also  acts 
in  the  same  way  on  blood  just  drawn.  Coagulation  is  also  hindered 
by    snake    poison    and    toxins    (see    page    143).     The   coagulation   may  be 

facilitated  by  raising  the  temperature;  by  contact  with  foreign  bodies,  i& 

which  the  blood  adheres;   by  stirring  or  beating  it;   by  admission  of  air; 


190  THE  BLOOD. 

bv  diluting  with  very  small  amounts  of  water;  by  the  addition  of  platinum- 
ISIack  or  finely  powdered  carbon ;  by  the  addition  of  laky  blood,  which  does 
not  act  bv  the  presence  of  dissolved  blood-coloring  matters,  but  by  the 
stromata  of  the  blood-corpuscles,  and  also  by  the  addition  of  the  leuco- 
cytes fmm  the  lymphatic  glands,  or  a  watery  saline  extract  of  the  lym- 
phatic glands,  testicles,  or  thymus  and  various  other  organs  (Delezenne, 
Wright,  Arthtjs  1  and  others). 

An  important  question  to  answer  is  why  the  blood  remains  fluid  in  the 
circulation,  while  it  quickly  coagulates  when  it  leaves  the  circulation.  The 
reason  why  blood  coagulates  on  leaving  the  body  is  therefore  to  be  sought 
for  in  the  influence  which  the  walls  of  the  living  and  uninjured  blood-vessels 
exert  upon  it.  These  views  are  derived  from  the  observations  of  many 
investigators.  From  the  observations  of  Hewsox,  Lister,  and  Fredericq 
it  is  known  that  when  a  vein  full  of  blood  is  ligatured  at  the  two  ends  and 
removed  from  the  body,  the  blood  may  remain  fluid  for  a  long  time. 
Brucke  L'  allowed  the  heart  removed  from  a  tortoise  to  beat  at  0°  C,  and 
found  that  the  blood  remained  uncoagulated  for  some  days.  The  blood 
from  another  heart  quickly  coagulated  when  collected  over  mercury.  In  a 
dead  heart,  as  also  in  a  dead  blood-vessel,  the  blood  soon  coagulates,  and 
also  when  the  wails  of  the  vessel  are  changed  by  pathological  processes. 

What  then  is  the  influence  which  the  walls  of  the  vessels  exert  on  the 
liquidity  of  the  circulating  blood?  Freuxd  has  found  that  the  blood 
remains  fluid  when  collected  by  means  of  a  greased  canula  under  oil  or  in  a 
vessel  smeared  with  vaseline.  If  the  blood  collected  in  a  greased  vessel  be 
beaten  with  a  glass  rod  previously  oiled,  it  does  not  coagulate,  but  it  quickby 
coagulates  on  beating  it  with  an  unoiled  glass  rod  or  when  it  is  poured 
into  a  vessel  not  greased.  The  non-coagulability  of  blood  collected  under 
oil  has  been  confirmed  later  by  Haycraft  and  Carlier.  Freund  found 
on  further  investigation  that  the  evaporation  of  the  upper  layers  of  blood 
or  their  contamination  with  small  quantities  of  dust  causes  a  coagulation 
even  in  a  vessel  treated  with  vaseline.  According:  to  Freund,3  it  is  this 
adhesion  between  the  blood  or.  as  the  blood  shows  an  adhesion  to  the 
normal  vessel  walls  (Beno  Levy)?  between  its  form  elements  and  a  foreign 
substance — and  the  diseased  walls  of  the  vessel  also  act  as  such — that  gives 
the  impulse  towards  coagulation,  while  the  lack  of  adhesion  prevents  the 
blood  from  coagulating.     This  adhesion  of  the  form-elements  of  the  blood 

1  Delezenne,  Arch,  de  Physiol.  (5),  8;  Wright,  Journ.  of  Physiol.,  28;  Arthus- 
Journ.  de  Physiol,  et  Pathol.,  4. 

2He\vson's  works,  edited  by  Gulliver,  London,  1876.  Cited  from  Gamgee,  Text, 
book  of  Physiol.  Chem.,  1,  1880.  Lister,  cited  from  Gamgee,  ibid.;  Fredericq,  "Re- 
cherches  sur  la  constitution  du  plasma  sanguin,"  Gand,  1878;  Brucke,  Virchow's 
Arch.,  12. 

3  Freund,  Wien.  med.  Jahrb.,  1886;  Haycraft  and  Carlier,  Journ.  of  Anat.  and 
Physiol.,  22;   Beno  Levy,  Arch.  f.  (Anat.  u.)  Physiol.,  1899.     Supplbd. 


COAGULATION  OF  THE  BLOOD.  l'Jl 

to  certain  foreign  substances  seems  to.  induce  changes  which  apparently 
stand  in  a  certain  relationship  to  the  coagulation  of  the  blood. 

The  views  in  regard  to  these  changes  are,  unfortunately,  very  contra- 
dictory. According  to  Alex.  Schmidt  '  and  the  Dorpat  eh  BOOL  an 
abundant  destruction  of  the  leucocytes  takes  place  in  coagulation  and 
important  constituents  for  the  coagulation  of  the  fibrin  pass  into  the  plasma. 
According  toother  experimenters  the  essential  is  not  a  destruction  of  the 
leucocytes,  but  an  elimination  of  constituents  from  the  cell-  into  the  plasma. 
This  process  is  called  plasmoschisis  by  Lowit.2  The  question  whether  the 
cell-body  (Giesbach)  or  the  nucleus  (Lilienfeld  3)  takes  part  in  this 
process  remains  for  the  present  undecided,  but  it  seems  positively  proven 
that  the  leucocytes  have  a  certain  relationship  to  the  coagulation.  Great 
importance  has  been  ascribed  to  the  blood  platelets  in  coagulation,  as  certain 
investigators  (Bizzozero,  Lilienfeld,  Schwalbe)  have  found  that  they 
cause  or  accelerate  coagulation,  while  others  (Petrone)  on  the  contrary 
find  a  retarding  action.4 

Wooldridge5  takes  a  very  peculiar  position  in  regard  to  this  question: 
he  considers  the  form-elements  as  only  of  secondary  importance  in  coagulation. 
As  found  by  him,  a  peptone-plasma  which  has  been  freed  from  all  form-con- 
stituents by  means  of  centrifugal  force  yields  abundant  fibrin  when  it  is  not 
separated  from  a  substance  which  precipitates  on  cooling.  This  substance, 
which  Wooldridge  has  called  A-fibrinogen,  seems  to  all  appearances  to  be  a 
nucleoproteid,  which,  according  to  the  unanimous  viewr  of  several  investigators, 
originates  from  the  form-elements  of  the  blood,  either  the  blood-plates  or  the 
leucocytes,  and  the  generally  accepted  view  as  to  the  great  importance  of  the 
form-elements  in  the  coagulation  of  the  blood  is  not  really  contrary  to  Wooii- 
dridge's  experiments. 

The  views  arc  greatly  divided  in  regard  to  those  bodies  which  are  elim- 
inated from  the  form-elements  of  the  blood  before  and  during  coagulation. 

According  to  Alex.  Schmidt  the  leucocytes,  like  all  cells,  contain  two 
chief  groups  of  constituents,  one  of  which  accelerates  coagulation,  while  the 
other  retards  or  hinders  it.  The  first  may  be  extracted  from  the  cells  by 
alcohol,  while  the  other  cannot  be  extracted.     Blood-plasma  contains  only 

1  Pfliiger's  Arch.,  11.  The  works  of  Alex.  Schmidt  are  found  in  Arch.  f.  Anat. 
und  Physiol.,  1861,  1862;  Pfliiger's  Arch.,  6,  9,  11,  13.  See  especially  Alex.  Schmidt, 
Zur  Blutlehre  (Leipzig,  1892),  which  also  gives  the  work  of  his  pupils,  and  Weitere 
Beitrage  zur  Blutlehre,  1895. 

2  Wien.  Sitzungsber.,  S9  and  90,  and  Prager  med.  Wochenschr. ,  1889.  Referred 
to  in  Centralbl.  f.  d.  med.  Wissensch.,  28,  265. 

s  Giesbach.  Pfliiger's  Arch.,  50,  and  Centralbl.  f.  d.  med.  Wissensch.,  1892;  Lilien- 
feld, Ueber  Leukocyten  und  Blutgerinnung,  Yerhandl.  d.  physiol.  Gesellsoh.  zu  Berlin, 
No.  11,  1892;  Ueber  den  flussigen  Zustand  des  Blutes,  etc.,  Und.,  No.  16,  1892;  and 
Weitere  Beitrage  zur  Kenntnisse  der  Blutgerinnung,  ibid.,  July,  1SS3.  Zeitschr.  f. 
physiol.  Chem.,  20. 

*  See  Mary's  Jahresber.,  31,  170;  Schwalbe,  Unters.  zu  Blutgerinnung,  etc., 
Braunschweig,  1900. 

6  Die  Gerinnung  des  Blutes  (published  by  M.  v.  Prey,  Leipzig,  1891). 


192  THE  BLOOD. 

traces  of  thrombin,  according  to  Schmidt,  but  does  contain  its  antecedent, 
prothrombin.  The  bodies  which  accelerate  coagulation  are  neither  thrombin 
nor  prothrombin,  but  they  act  in  this  wise  in  that  they  split  off  thrombin 
from  the  prothrombin.  On  this  account  they  are  called  zymoplastia  sub- 
stances by  Alex.  Schmidt.  The  nature  of  these  bodies  is  unknown,  and 
Schmidt  has  given  no  notice  of  their  behavior  with  the  lime  salts,  which  have 
been  found  to  have  zymoplastic  activity  by  other  investigators. 

The  constituents  of  the  cells  which  hinder  coagulation  and  which  are 
insoluble  in  alcohol-ether  are  compound  proteids  and  have  been  called 
cytoglobin  and  preglobulin  by  Schmidt.  The  retarding  action  of  these 
bodies  may  be  suppressed  by  the  addition  of  zymoplastic  substances,  and 
the  yield  of  fibrin  on  coagulation  in  this  case  is  much  greater  than  in  the 
absence  of  the  compound  proteid  retarding  coagulation.  This  last  supplies 
the  material  from  which  the  fibrin  is  produced.  The  process  is,  according 
to  Schmidt,  as  follows:  The  preglobulin  first  splits,  yielding  serglobulin, 
then  from  this  the  fibrinogen  is  derived  and  from  this  latter  the  fibrin  is 
produced.  The  object  of  the  thrombin  is  twofold.  The  thrombin  first  splits 
the  fibrinogen  from  the  paraglobulin  and  then  converts  the  fibrinogen 
into  fibrin.  The  assumption  that  fibrinogen  can  be  split  from  paraglobulin 
has  not  sufficient  foundation  and  is  even  improbable. 

According  to  Schmidt  the  retarding  action  of  the  cells  is  prominent 
during  life,  while  the  accelerating  action  is  especially  pronounced  outside 
of  the  body  or  by  coming  in  contact  with  foreign  bodies.  The  parenchy- 
mous  masses  of  the  organs  and  tissues,  through  which  the  blood  flows  in 
the  capillaries,  are  those  cell-masses  which  serve  to  keep  the  blood  fluid 
during  life. 

Lilienfeld  has  given  further  proof  as  to  the  occurrence  in  the  form- 
elements  of  the  blood  of  bodies  which  accelerate  or  retard  the  coagulation. 
According  to  this  author  the  nature  of  these  bodies  is  very  markedly  differ- 
ent from  Schmidt's  idea.  While,  according  to  Schmidt,  the  coagulation- 
accelerators  are  bodies  soluble  in  alcohol,  and  the  compound  proteids  ex- 
hausted with  alcohol  only  act  retardingly  on  coagulation,  Lilienfeld  states 
that  the  substance  which  acts  acceleratingly  and  retardingly  on  coagulation 
consists  of  a  nucleoproteid,  namely,  nucleohiston.  Nucleohiston  readily 
splits  into  leuconuclein  and  histon,  the  first  of  which  acts  as  a  coagulation- 
excitant,  while  the  other,  introduced  into  the  blood-vascular  system,  either 
intravascular  or  extravascular,  robs  the  blood  of  its  property  of  coagulating. 
Introduced  into  the  circulatory  system  the  nucleohiston  splits  into  its  two 
components.  It  therefore  causes  extensive  coagulation  on  one  side  and 
makes  the  remainder  of  the  blood  uncoagulable  on  the  other.  This  theory 
as  well  as  that  of  Schmidt  is  not  based  upon  sufficiently  positive  facts. 

Brucke  showed  long  ago  that  fibrin  left  an  ash  containing  calcium 
phosphate.    The  fact  that  calcium  salts  may    facilitate  or  even  cause  a 


COAGULATION  OF   THE  BLOOD.  193 

coagulation  in  liquids  poor  in  ferment  has  been  known  for  BeveraJ  Tears 

through  the  researches  of  HAMMARSTEN,  GREEN,  RlNGER,  ami  SaINSFDBT. 
The  necessity  of  the  lime  salts  for  the  coagulation  of  blood  and  plasma  was 
first  shown  positively  by  the  important  investigations  of  Annus  and 
Pages.  RecenJ  investigations  of  Sabbatani  '  have  also  shown  the  impor- 
tance of  calcium  salts  or  the  free  calcium  ions  for  <  oagulation  without 
explaining  the  mode  of  their  action. 

According  to  the  generally  accepted  view  of  Arthus  and  Pages  the  soluble 
lime  salts  precipitable  by  oxalate  are  necessary  requisites  for  the  fermentive 
transformation  of  fibrinogen  because  thrombin  remains  inactive  in  the  absence 
of  soluble  lime  salts.  This  view  is  untenable,  as  shown  by  the  researches  of 
Ai.i.x.  Schmidt,  Pekeui.uung,  and  Hammarsten.2  Thrombin  acts  as  well  in 
the  absence  as  in  the  presence  of  precipitable  lime  salts. 

Liiibnpeld's  t  eory  that  the  leuconuclein  splits  <  ff  a  protein  substance, 
(kromboain,  from  the  fibrinogen,  and  this  thromboein  forms  an  insoluble  combina- 
tion with  the  lime  present,  producing  thrombosin  lime  (fibrin),  which  separates,  is 
incorrect  according  to  Hammarsten,  Schafer,  and  Cramer.1  Lilienfeld's 
thrombosin  is  nothing  but  fibiinogen  which  is  precipitated  by  a  lime  salt  from  a 
salt -poor  or  salt-free  solution. 

According  to  Pekelharixg  *  thrombin  is  the  lime  combination  of  pro- 
thrombin, and  the  process  of  coagulation  consists,  according  to  him,  in 
the  thrombin  transferring  the  lime  to  the  fibrinogen,  which  is  hereby  con- 
verted into  an  insoluble  lime  combination,  fibrin.  Among  the  objections 
to  this  theory  can  be  mentioned,  among  others,  the  fact  that  fibrin  has  been 
obtained  not  absolutely  free  from  lime,  but  still  so  poor  in  lime  (Hammar- 
stex  5)  that  if  the  lime  belongs  to  the  fibrin  molecule  it  must  be  more 
than  ten  times  greater  than  the  haemoglobin  molecule,  which  is  not  prob- 
able. These  as  well  as  many  other  observations  decide  that  the  lime  is 
carried  down  by  the  fibrinogen  only  as  a  contamination. 

If,  as  it  seems,  the  lime  is  not  of  importance  in  the  transformation  of 
fibrinogen  into  fibrin  in  the  presence  of  thrombin,  still  this  does  not  con- 
tradict the  above-mentioned  observations  of  Arthus  and  Pages  that  the 
lime  salts  are  necessary  for  the  coagulation  of  blood  and  plasma.  It  is  very. 
probable  that  the  lime  salts,  as  admitted  by  Pekelharixg,  are  a  necessary 
requisite  for  the  transformation  of  prothrombin  into  thrombin. 

1  Hammarsten,  Nova  Acta  reg.  Soc.  Scient.  Upsal  (3),  10,  1879;  Green,  Journ.  of 
Physiol.,  8;  Ringer  and  Sainsbury,  ibid.,  11  and  12;  Arthus  et  Pages  and  Arthus, 
see  foot-note  4,  page  143;  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22;  Sabbatani, 
cited,  Centralbl.  f.  Physiol.,  16,  665. 

2  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22,  where  the  other  investigators  are 
cited. 

3  Hammarsten,  1.  c. ;  Schafer,  Journ.  of  Physiol.,  17;  Cramer,  Zeitschr.  f.  physiol. 
Chem.,  23. 

4  See  foot-note  5,  page  146,  and  especially  Yirchow's  Festschrift,  1,  1S91. 
6  Zeitschr.  f.  physiol.  Chem.,  28. 


194  THE   BLOOD. 

It  is  a  question  whether  the  prothrombin  exists  in  the  plasma  of  the 
circulating'  blood  or  whether  it  is  a  body  eliminated  from  the  form-elements- 
before  coagulation.  Alex.  Schmidt  claims  that  the  circulating  plasma  con- 
tains prothrombin,  but  Pekelharing  disclaims  this.  Blood-plasma  ob- 
tained by  means  of  leech  infusion  does  not  coagulate  on  the  addition  of 
lime  salts,  but  does  on  the  addition  of  a  prothrombin  solution.  The  form- 
elements,  especially  the  blood-plates,  are  particularly  well  preserved  in 
such  plasma;  and  according  to  Pekelharing  it  is  probable  that  the  cir- 
culating plasma  does  not  contain  any  mentionable  amounts  of  prothrombin,, 
and  that  this  body  emerges  from  the  form-elements,  perhaps  the  blood- 
plates,  before  coagulation.  The  difference  between  the  views  of  Schmidt 
and  Pekelharing  on  this  point  is  as  follows:  According  to  Schmidt  it  is 
the  zymoplastic  substances  which  pass  from  the  form-elements  into  the 
plasma  and  transform  the  prothrombin  existing  preformed  therein.  Pekel- 
haring claims  that  it  is  the  prothrombin  which  passes  from  the  form- 
elements  into  the  plasma  and  is  converted  into  thrombin  by  the  lime  salts 
of  the  plasma.  The  fact  that  sodium-fluoride  plasma  contains  no  pro- 
thrombin shows  that  the  plasma  of  circulating  blood  does  not  contain  any 
prothrombin  ( Arthtjs)  . 

As  Alex.  Schmidt  has  shown  that  blood-serum  whose  power  of  excit- 
ing coagulation  has  been  considerably  reduced  by  standing  in  the  air,  can 
be  activated  again  by  alkali,  Morawitz  *  found  that  such  a  reactivation 
can  be  brought  about  by  acids  and  by  alcohol  and  that  the  thrombin  formed 
in  this  reactivation  is  /^-thrombin,  which  is  different  from  the  a-thrombin 
of  blood-serum.  These  two  thrombins  correspond  to  two  prothrombins. 
The  a-prothrombin  which  occurs  in  oxalate  plasma  is  changed  into- 
a-thrombin  by  lime  salts.  The  a-prothrombin  does  not  exist  either  in  fresh 
or  in  old  serum;  it  occurs  as  above  stated  in  the  oxalate  plasma,  but  not  in 
the  fluoride  plasma.  Blood-serum  always  contains  traces  of  one  pro- 
thrombin, the  /3-pro thrombin,  which  does  not  occur  in  the  plasma,  but  is 
first  produced  in  coagulation  from  an  unknown  substance  by  the  action 
of  the  lime  salts. 

As  the  /^-thrombin  is  not  formed  in  the  coagulation,  according  to  Mora- 
witz, and  as  it  does  not  occur  either  in  the  plasma  or  in  the  serum,  it  is. 
evident  that  it  has  nothing  to  do  with  the  ordinary  coagulation  of  the 
blood  and  hence  can  be  ignored.  According  to  the  later  communication 
of  Morawitz  2  the  fibrin  ferment  active  in  the  coagulation  of  the  blood  is 
produced  by  the  combined  action  of  at  least  three  substances:  (a)  the 
thromborjen  (prothrombin  in  the  ordinary  sense),  (b)  the  thrombokinase 
(zymoplastic  substance),  and  (c)  the  lime  salts.  The  circulating  plasma 
contains  either  thrombogen  or  thrombokinase,  and  the  last-mentioned  body 

1  Hofmeister's  Beitrage,  4. 

2  Deutsch.  Arch.  f.  klin.  Med.,  79. 


COAGULATIOX  OF   THE   BLOOD.  195 

is  secreted  in  the  plasma  by  the  form-elementa  of  the  blood.  The  throm- 
bogen  originates,  according  to  MoRAWTTZ,  from  the  blood-plates.  The 
production  of  the  zymoplastic  substance  or  thrombokinase  is  a  general 
property  of  protoplasm  and  hence  of  the  leucocytes.  This  investigator 
has  also  found  that  a  body  can  be  obtained  from  oxalate  and  fluoride 
plasma  which  has  a  retarding  influence  upon  the  action  of  thrombin  depend- 
ing upon  its  quantity,  hence  it  has  been  called  antithromhin. 

Independent  of  Morawitz,  Fuld  l  has  arrived  in  the  main  at  similar 
results,  although  from  the  short  communication  there  seems  to  lie  some 
contradiction.  He  admits  the  necessity  of  three  things  for  the  produc- 
tion of  fibrin  ferment,  namely,  lime  salts,  proferment,  and  zymoplastic 
substance,  which  latter  originates  from  the  various  form-elements.  The 
three  bodies,  proferment,  zymoplastic  substance,  and  the  complete,  active 
ferment,  he  calls  plasmozym,  cytozym,  and  holozym  respectively.  Contrary 
to  Morawitz's  view  that  the  fluoride  plasma  is  free  from  a-prothrombin, 
Fuld  claims  that  it  contains  plasmozym.  The  same  is  true  for  the  natural 
blood-plasma.  The  reason  why  the  living  blood  remains  fluid  is,  according 
to  Fuld,  chiefly  due  to  the  fact  that  the  cytozym  is  always  formed  only 
slowly  and  that  the  ferment  produced  is  quickly  converted  into  an  inactive 
form,  and  also  because  the  blood  contains  an  anti-body  for  the  thrombin. 
The  production  of  thrombin  and  /3-prothrombin  (the  latter  called  metazym 
by  him)  Fuld  explains  in  another  manner.  By  the  combined  action  of 
the  three  bodies,  plasmozym,  cytozym,  and  lime  salts,  the  holozym 
(  =  a-thrombin)  is  produced.  This  latter  may  be  converted  into  metazym 
(^-prothrombin),  from  which  the  neozym  (=/3-thrombin)  is  then  formed 
by  the  action  of  alkalies  or  acids. 

In  regard  to  the  much-contradicted  role  of  the  form-elements  of  the 
blood  in  coagulation  we  have  the  recent  investigations,  especially  of  Arti  [US 
and  of  Dastre.2  According  to  Arthus  the  fibrin  ferment  is  not  a  product 
of  the  death  of  the  cells,  but  a  secretion  product.  This  secretion  is  pre- 
vented by  sodium  fluoride,  hence  fluoride  plasma  contains  neither  thrombin 
nor  prothrombin.  This  secretory  activity  of  the  cells  is  increased  on 
contact  with  solids  or  by  the  action  of  tissue  fluids.  Dastre,3  who  with 
his  pupils  opposes  the  view  as  to  a  destruction  of  leucocytes  in  coagulation, 
considers  also  that  the  ferment  is  not  a  product  of  the  death  of  the  cell, 
but  is  a  constituent  expelled  from  the  living  cell  under  the  influence  of 
osmotic  conditions. 

From  the  above  discussion  it  is  evident  that  there  is  at  the  present  no  gen- 
erally accepted  theory  based  upon  positive  observations,  of  the  extravas- 
cular  coagulation  of  blood. 

1  Centralbl.  f.  Physiol.,  17,  529. 
aCompt.  rend,  de  la  soc.  biolog.,  55. 
3  Ibid. 


196  THE  BLOOD. 

Intravascular  coagulatiaQ.  It  has  been  shown  by  Alex.  Schmidt  and 
his  students,  as  also  by  Wooldridge,  Wright,1  and  others,  that  an  intra- 
vascular coagulation  may  be  brought  about  by  the  intravenous  injection 
into  the  circulating  blood  of  a  large  quantity  of  a  thrombin  solution,  as 
also  by  the  injection  of  leucocytes  or  tissue  fibrinogen  (impure  nucleopro- 
teid).  Intravascular  coagulation  may  be  brought  about  also  under  other 
conditions,  such  as  after  the  injection  of  snake-poison  (Martin  2  and  others) 
or  certain  of  the  proteid-like  colloid  substances,  synthetically  prepared 
according  to  Grimaux  's  method  (Halliburton  and  Pickering  3) .  If  too- 
little  of  the  above-mentioned  bodies  be  injected,  then  we  observe  only  a 
marked  retarding  tendency  in  the  coagulation  of  the  blood.  According  to 
Wooldridge  it  can  generally  be  maintained  that  after  a  short  stage  of 
accelerated  coagulability,  which  may  lead  to  a  total  or  partial  intravascular 
coagulation,  a  second  stage  of  a  diminished  or  even  arrested  coagulability 
of  the  blood  follows.  The  first  stage  is  designated  (Wooldridge)  as  the 
positive  and  the  other  as  the  negative  phase  of  coagulation.  These  state- 
ments have  been  confirmed  by  several  investigators. 

There  is  no  doubt  that  the  positive  phase  is  brought  about  by  an  abun- 
dant introduction  of  thrombin,  or  by  a  rapid  and  abundant  formation  of 
the  same.  According  to  Alex.  Schmidt,  the  zymoplastic  substances- 
soluble  in  alcohol  are  active  in  these  processes,  while  according  to  the 
investigations  of  Lilienfeld  this  action  is  caused  more  likely  by  the  leuco- 
nucleins  split  off  from  the  nucleohiston.  According  to  Wooldridge,  his 
tissue  fibrinogen  does  not  produce  any  intravascular  coagulation  if  it  is  freed 
from  contaminating  bodies  by  means  of  alcohol.  This  corresponds  to  the 
statements  of  Alex.  Schmidt. 

The  explanation  of  the  production  of  the  negative  phase,  which  can 
easily  be  produced  by  proteoses,  by  various  bodies,  such  as  extracts  of 
organs,  eel-serum,  enzymes,  bacterial  toxins,  snake-poisons,  etc.,  has  been 
attempted  in  different  ways.  Lilienfeld  seeks  the  reason  in  a  cleavage  of 
histon,  which  has  a  retarding  action,  from  the  nucleohiston.  The  retard- 
ing action  of  histon  has  been  shown,  but  not  its  cleavage  from  nucleohiston 
in  this  process.  According  to  Wright  and  Pekelharing,  the  retarding 
substances  are  proteoses,  which  are  formed  in  the  decomposition  of  the 
injected  nucleopro teids.  In  opposition  to  this  view  there  is  the  fact  that 
other  investigators,  as  Halliburton  and  Brodie,*  have  been  unable  to 

1  A  Study  of  the  Intravascular  Coagulation,  etc.,  Proceed,  of  the  Roy.  Irish  Acad. 
(3),  2.  See  also  Wright,  Lecture  on  Tissue  or  Cell  Fibrinogen,  The  Lancet,  1892; 
and  Wooldridge's  Method  of  Producing  Immunity,  etc.,  Brit.  Med.  Journal,  Sept.,  1891. 

2  Journ.  of  Physiol. ,  15. 

3  Ibid.,  18. 

4  Wright,  1.  c.j  Lilienfeld,  1.  0.5  Pekelharing,  1.  C.J  Halliburton  and  Brodie,  Journ. 
of  Physiol.,  17. 


INTRAVASCl  J. Ah'  COAGl  LATION.  11  i 

detect  any  proteose  in  the  blood  or  urine  under  these  conditions.  The 
retarding  action  of  the  poisonous  substance  of  snake-blood,  which  is  not  a 
nucleoproteid,  as  well  as  the  action  of  proteoses,  speak  against  the  as- 
sumption as  to  a  retarding  decomposition  product  of  the  injected  nucleo- 
proteid. 

According  to  the  observations  of  Faxo  and  Schmidt-Mi' liikim.1  which 
have  been  substantiated  and  enlarged  upon  by  other  observers,  the  blood 
of  an  animal  loses  its  power  of  coagulation  after  an  injection  of  a  suffi- 
ciently large  quantity  of  proteose  and  after  a  time  regains  its  power  or 
coagulation.  A  new  injection  of  proteose  is  now  inactive,  if  too  long  a 
time  does  not  intervene  between  the  two  injections,  and  the  animal  is  now 
immune  against  proteoses.  This  action  does  not  depend,  as  Pick  and  Spiro 
have  shown,  upon  the  proteose  itself,  but  upon  a  contaminating  substance, 
the  peptozym.  Peptozym-free  proteose  having  no  action  upon  coagula- 
tion can  be  obtained  from  certain  proteids,  while  peptozym  can  be  obtained 
free  from  proteoses  and  peptones  from  several  organs.  Coagulation- 
retarding  substances  may  also,  as  Coxradi  2  found,  be  produced  in  the 
autolysis  of  organs;  but  they  are  different  from  the  peptozym,  which 
differs  from  the  first  by  being  inactive  on  the  blood  in  vitro  and  only  having 
a  retarding  action  upon  coagulation  when  introduced  into  the  blood  circu- 
lation. 

We  have  a  large  number  of  researches  on  the  action  of  proteoses  and 
of  other  retarding  substances  by  different  investigators,  such  as  Grosjeax, 
Ledoux,  Coxtejeax,  Dastre,  Floresco,  Athaxasiu,  Carvallo,  Glky. 
Pachox,  Nolf,  Spiro  and  Ellixger,  and  others,  but  those  of  Delezexm:  3 
are  of  the  greatest  importance.  It  seems  probable  from  these  investi- 
gations that  the  leucocytes  and  the  liver  are  of  great  importance  in 
these  processes.  The  view  which  coincides  best  with  the  facts  seems  to 
be  Delezexxe's.  According  to  him  the  facts  can  be  simplest  explained 
by  the  questionable  body  producing  a  destruction  of  the  leucocytes  and 
that  here  a  substance  accelerating  coagulation  and  another  having  a  retard- 
ing action  are  set  free.  The  first  is  destroyed  by  the  liver  and  hence  the 
action  of  the  retarding  substances  is  obtained. 


^ano,  Arch.  f.  (Anat.  u.)  Physiol.,  1881;  Schmidt-Mulheim,  ibid.,  1880. 

1  Pick  and  Spiro,  Zeitschr.  f.  physiol.  Chem.,  31;  Conradi,  Hofmeister's  Beitnige,  1. 
See  also  Underhill,  Amer.  Journ.  of  Physiol.,  9. 

'  Grosjean,  Travaux  du  laboratoire  de  L.  Fredericq,  4,  Liege,  1S92;  Ledoux,  ibid., 
6,  189G;  Noll,  Bull.  l'Acad.  roy.  de  Belgique,  1902;  Spiro  and  Ellinger,  Zeitschr.  f. 
physiol.  Chem.,  23.  The  works  of  the  above-mentioned  French  investigators  can  lie 
found  in  Compt.  rend.  soc.  biol.,  46,  47,  48,  50,  and  51,  and  Arch.  d.  Physiol.  (5  .  7 
8,  9,  and  10;  see  also  especially  Delezenne,  Arch.  d.  Physiol.  (5),  10;  Compt.  rend, 
soc.  biol.,  51,  and  Compt.  rend.,  130. 


198  THE  BLOOD. 

In  regard  to  the  coagulation  of  the  blood  of  invertebrates  we  refer  to 
the  recent  investigations  of  Ducceschi  and  of  Loeb.1 

The  gases  of  the  blood  will  be  treated  in  Chapter  XVII  (on  respira- 
tion). 

IV.     The  Quantitative  Composition  of  the  Blood. 

The  quantitative  analyses  of  blood  are  of  little  value.  We  must  ascer- 
tain on  one  side  the  relationship  of  the  plasma  and  blood-corpuscles  to  each 
other,  and  on  the  other  side  the  constitution  of  each  of  these  two  chief 
constituents.  The  difficulties  which  stand  in  the  way  of  such  a  task, 
especially  in  regard  to  the  living,  non-coagulated  blood,  have  not  been 
removed.  Since  the  constitution  of  the  blood  may  differ  not  only  in  differ- 
ent vascular  regions,  but  also  in  the  same  region  under  different  circum- 
stances, which  renders  also  a  number  of  blood  analyses  necessary,  it  can 
hardly  appear  remarkable  that  our  knowledge  of  the  constitution  of  the 
blood  is  still  relatively  limited. 

The  relative  volume  of  blood-corpuscles  and  serum  in  defibrinated  blood 
may  be  determined,  according  to  L.  and  M.  Bleibtreu,2  by  various 
methods  if  the  defibrinated  blood  is  mixed  with  different  proportions  of 
isotomic  NaCl  solution  (1  vol.  of  the  blood  to  at  least  1  vol.  salt  solution), 
the  blood-corpuscles  allowed  to  settle  to  the  bottom  or  facilitated  by  cen- 
trifugal force,  and  the  clear  supernatant  mixture  of  serum  and  common- 
salt  solution  siphoned  off.    The  methods  are  as  follows: 

1.  Determine  the  quantity  of  nitrogen  in  at  least  two  different  portions  of 
the  mixture  of  serum  and  salt  solution  by  means  of  Kjeldahl's  method  and 
calculate  the  quantity  of  proteid  corresponding  thereto  by  multiplying  with 
6.25,  and  the  relative  volume  of  blood  x,  and  also  the  volume  of  the  structural 
elements  (l—x),  are  found  by  the  following  equation: 

(   -  \  -h.   _fi 

°2  °1 

In  this  equation  (for  mixtures  1  and  2)  bt  or  b2  represents  the  volume  of  blood 
in  the  mixture,  s1  or  s2  the  volume  of  salt  solution,  and  ex  or  e2  the  quantity  of 
proteid  in  a  certain  volume  of  each  mixture. 

2.  Determine  the  specific  gravity  of  the  blood-serum,  of  the  salt  solutions,  and  of 
at  least  one  of  the  mixtures  of  serum  and  salt  solution  by  means  of  a  pyknometer. 
The  relative  volume  of  serum  x  is  found  in  this  following  equation: 

±    S~K 

X~  b  '  S0-K' 

In  this  equation  s  and  b  represent  the  volumes  of  salt  solution  and  blood  mixed. 
S  represents  the  specific  gravity  of  the  serum  and  salt  solution  obtained  on 
allowing  the  blood-corpuscles  to  settle,  Sa  the  specific  gravity  of  the  serum,  and 
K  that  of  the  salt  solution. 

For  horse's  blood  two  other  shorter  methods  may  be  made  use  of  (see  the 
original  article). 

■  x  Ducceschi,  Hofmeister's  Beitriige,  3;  Loeb,  Biological  Bulletin,  4,  1903. 
2  Pfliiger's  Arch.,  51,  55,  and  60. 


QUANTITATIVE  BLOOD  ANALYSTS.  109 

Important  objections  have  been  presented  by  several  investigators,  such 
as  Kykman,  BlEBNACKI,  and  Hedin,1  against  the  above  methods,  whose 
value,  therefore,  is  questionable.  The  same  is  also  true  for  another  method, 
suggested  by  St.  BuOABSKY  and  TANGL  and  partly  corrected  by  Stewart 
in  regard  td  the  calculations,  which  is  based  upon  a  difference  in  electric 
conductivity  for  the  blood  and  the  plasma.  STEWART  "  has  also  worked 
out  a  colorimetric  method  for  the  estimation  of  the  volume  of  the  blood- 
corpuscles  and  the  plasma,  which  seems  to  be  worth  applying. 

For  clinical  purposes  the  relative  volume  of  corpuscles  in  the  blood  may 
be  determined  by  the  use  of  a  small  centrifuge  called  hcematocrit,  constructed 
by  Blix  and  described  and  tested  by  Hedin.  A  measured  quantity  of 
blood  is  mixed  with  a  known  volume  (best  an  equal  volume)  of  a  fluid 
which  prevents  coagulation.  This  mixture  is  introduced  into  a  tube  and 
then  centrifuged.  According  to  Hedin  it  is  best  to  treat  the  blood,  which 
is  kept  fluid  by  1  p.  m.  oxalate,  with  an  equal  volume  of  a  9  p.  m.  NaG 
solution.  After  complete  centrifugalization  the  layer  of  blood-corpuscles  is 
read  off  on  the  graduated  tube  and  the  volume  of  blood-corpuscles  (or  more 
correctly  the  layer  of  blood-corpuscles)  calculated  in  100  vols,  of  the  blood 
therefrom.  By  means  of  comparative  counts  Hedin  and  Daland  have 
found  that  an  approximately  constant  relation  exists  between  the  volume 
of  the  layer  of  blood-corpuscles  and  the  number  of  red  corpuscles  under 
physiological  conditions,  so  that  the  number  of  corpuscles  may  be  calculated 
from  the  volume.  Daland  3  has  shown  that  such  a  calculation  gives 
approximate  results  also  in  disease  when  the  size  of  the  blood-corpuscles 
does  not  essentially  deviate  from  the  normal.  In  certain  diseases,  such  as 
pernicious  ansemia,  this  method  gives  such  inaccurate  results  that  it  cannot 
be  used. 

In  determining  the  relationship  between  the  weight  of  blood-corpuscles 
and  the  weight  of  blood-fluid,  we  generally  proceed  in  the  following  manner: 

If  any  substance  is  found  in  the  blood  which  belongs  exclusively  to  the 
plasma  and  does  not  occur  in  the  blood-corpuscles,  then  the  amount  of 
plasma  contained  in  the  blood  may  be  calculated  if  we  determine  the  amount 
of  this  substance  in  100  parts  of  the  plasma  or  serum,  respectively,  on  one 
side  and  in  100  parts  of  the  blood  on  the  other.  If  we  represent  the  amount 
of  this  substance  in  the  plasma  by  p  and  in  the  blood  by  b,  then  the  amount 

of  x  in  the  plasma  from  100  parts  of  blood  is  x=  — -^— . 

P 

Such  a  substance,  which  occurs  only  in  the  plasma,  is  fibrin  according 

to  Hoppe-Seyler,  sodium  according  to  Bunge  (in  certain  kinds  of  blood), 
and  sugar  according  to  Otto.4  The  experimenters  just  named  have  tried 
to  determine  the  amount  of  the  plasma  and  blood-corpuscles,  respectively, 
in  different  kinds  of  blood,  starting  from  the  above-mentioned  substances. 
Another  method  suggested  by  Hoppe-Seyler  is  to  determine  the  total 

1  Biemacki,  Zeitschr.  f.  physiol.  Chem.,  19;  Eykman,  Pfliiger's  Arch.,  60;  Hedin, 
ibid.,  and  Skand.  Arch.  f.  Physiol.,  5. 

1  Bugarsky  and  Tangl,  Centralbl.  f.  Physiol.,  11;  Stewart,  Journ.  of  Physiol.,  24. 

'Hedin,  Skand.  Arch.  f.  Physiol.,  2,  13-4  and  361,  and  5;  Pfliiger's  Arch.,  60; 
Daland,  Fortschritte  d.  Med.,  9. 

*  Hoppe-Seyler,  Ilandb.  d.  physiol.  u.  path.  chem.  Analyse,  6.  Aufl.;  Bunge,  Zeit- 
schr. f.  Biologie,  12;  Otto,  Pfliiger's  Arch.,  35. 


200 


THE  BLOOD. 


amount  of  haemoglobin  and  proteids  in  a  portion  of  blood,  and  on  the  other 
hand  the  amount  of  haemoglobin  and  proteids  in  the  blood-corpuscles  (from 
an  equal  portion  of  the  same  blood)  which  have  been  sufficiently  washed- 
with  common-salt  solution  by  centrifugal  force.  The  figures  obtained  as  a 
difference  between  these  two  determinations  corresponds  to  the  amount  of 
proteids  which  was  contained  in  the  serum  of  the  first  portion  of  blood. 
If  we  now  determine  the  proteids  in  a  special  portion  of  serum  of  the  same 
blood,  then  the  amount  of  serum  in  the  blood  is  easily  determined.  The 
usefulness  of  this  method  has  been  confirmed  by  Bunge  by  the  control 
experiments  with  the  sodium  determinations.  If  the  amount  of  serum  and 
blood-corpuscles  in  the  blood  is  known,  and  we  then  determine  the  amount 
of  the  different  blood-constituents  in  the  blood-serum  on  one  side  and  of 


Pig-blood. 

Ox-blood. 

Horse-blood. 

Dog-blood. 

Bull-blood. 

Sheep-blood. 

a 

,    3o 

0  oco 

0  0* 

3 

*°5 

3o 

(-US 
CO 

0 

■  [h 

T3  Cm 
OOM 
O  OCO 

3 

.us 

cu 

m 

0 
,  5» 

O    OCi 

0  oco 

3 

.CO 

S  oi 

SO 
CD 

m 

a 

.  |°° 

0  C* 
0  o-* 

3 

-CM 

En 

i.10 
<p 
W 

a 

0  oco 
0  oco 

3 

giO 

Geo 

cu 

m 

cu 
"o 

0  0  — 
0  oco 

3 

.00 
So 

poo 
fc.cO 

<D 

m 

272.20 

162.89 

142.20 

8.35 

0.213 
1.504 

0.027 
0.0455 

0.696 

0.0656 
0.642 
0.8956 
0.7194 

518.36 
46.54 

38.26 
0.684 
0.231 
0.805 
1.104 
0.448 

0.0123 

2.401 
0.152 

0.0689 

0.0233 

2.048 

0.1114 

0.0296 

192.65 

132.85 

103.10 

20.89 

1.100 
1.220 

0.0178 

0.7266 
0.2351 
0.544 

0.0056 
0.5901 
0.2392 
0.1140 

616.25 
58.249 

48.901 

0.708 

0.835 

1.129 

0.625 

0.0089 

2.9084 
0.1719 

0.0805 
0.0300 
2.4889 
0.1646 
0.0571 

243.87 
153.84 
125.8 
20.05 

0.26 
1.93 

0.02 
0.05 

1.32 
0.59 

0.04 
0.18 
0.98 
0.76 

551.14 
51.15 

42.65 
0.90 
0.31 
1.05 
0.50 
0.36 

0.01 

2.62 

0.15 

0.07 
0.03 
2.20 
0.15 
0.05 

277.71 
165.10 
145.6 
2.36 

0.56 
1.02 

0.05 

1.27 
0.11 
0.71 

0.03 
0.60 
0.67 
0.54 

514.30 
42.89 

34.05 
0.74 
0.37 
0.98 
0.91 
0.70 

0.01 

2.39 

0.14 

0.06 
0.03 
2.31 
0.14 
0.05 

206.81 

127.50 

106.40 

15.38 

0.610 
0.953 

0.0194 

0.839 
0.233 
0.562 

0.009 
0.628 
0.236 
0.133 

608.03 
57.66 

46.41 
0.679 
0.599 
1.244 
2.357 
0.494 

0.0089 

2.873 
0.174 

0.073 
0.027 
2.453 
0.156 
0.041 

200.39 

118.82 

102.80 

12.80 

1.147 
1.329 

0.0235 
0.760 
0.236 
0.545 

0.006 
0.575 
0.228 
0.088 

624.16 

56.63 

46.56 

0.708 

0.891 
1.088 

Fat 

0.859 

Phosphoric  acid  1 
as  nuclein          J 

0.4908 
0.0109 
2.917 

0.172 
0.089 

Magnesia 

0.027 
2.516 

Phosphoric  acid.  . 
Inorganic  P2O5.  .  . 

0.163 
0.057 

Water 

Solids 

Haemoglobin.  .  . 

Proteid 

Sugar 

Cholesterin.  .  .  . 

Lecithin 

Fat 

Fatty  acids .... 

Phosphoric  acid 
as  nuclein 

Soda 

Pota  b 

Iron  oxide 

Lime 

Magnesia 

Chlorine.  . 
Phosphoric  acid 
Inorganic  P2Oj. 


Goat-blood. 


0.028 

0.755 
0.236 
0.547 

0.014 
0.514 
0.243 
0.097 


592.54 
60.25 

50.96 
0.822 
0.698 
1.127 
0.0407 
0.398 

0.0117 

2.824 
0.160 

0.078 
0.020 
2.409 
0.154 
0.045 


—      1 


Cat-blood. 

Rabbit-blood. 

d 

CO 

0 

0 

-0 

3_ 

.05 

•B  &>* 

go 

-c  &oi 

Sfc 

0  occ 

30 

OON 

Ps3 

O  OCO 

« 

w 

m 

w 

270.90 

524.17 

235.74 

518-18 

163.11 

41.35 

136.37 

46.71 

143.2 

— 

123.50 

— 

11.62 

33.16 

4.55 

33.63 

— 

0.860 

— 

1.036 

0.556 

0.339 

0.268 

0.343 

1.354 

0.971 

1.722 

1.105 

0.446 

— 

0.749 

— 

0.282 

— 

0.507 

0.063 

0.009 

0.040 

0.015 

1.174 

2.512 

— 

2.789 

0.112 

0.148 

1.940 

0.162 

0.694 

— 

0.615 

— 

— 

0.062 

— 

0.072 

0.035 

0.024 

0.029 

0.028 

0.155 

2.360 

0.460 

2.438 

0.697 

0.133 

0.S35 

0.151 

0.515 

0.040 

0.645 

0.040 

Human  Blood, 
Man. 


349.09 
163.33 


Organic 
bodies 
159.59 


Inorg. 

3.74 

0.24 
1.59 


0.90 


300 


439.02 
47.96 


43.82 


4.14 

1.66 
0.15 


1.72 


Human  Blood, 
Woman. 


272.56 
123.68 


120.13 


3.55 

0.65 
1.41 


0.36 


£co 

30 


551.99 
51.77 


46.70 


5.07 

1.92 
0.20 


0.14 


QUANTITATIVE  BLOOD  ANALYSIS.  201 

the  total  blood  on  the  other,  the  distribution  of  these  different  blood-con- 
stituents in  the  two  chief  components  of  the  blood,  plasma  and  blood- 
corpuscles,  may  be  ascertained.  On  page  200  are  given  analyses  of  the 
blood  of  various  animals  by  Ahderhalden  x  according  to  Hoppe-Seyler's 
and  Bunge's  methods.  The  analyses  of  human  blood  by  C.  Schmidt  2  are 
older  and  were  made  according  to  another  method,  hence  perhaps  the 
results  for  the  weight  of  corpuscles  are  a  little  too  high.  All  the  results  are 
in  parts  per  1000  parts  of  blood. 

The  relation  between  blood-corpuscles  and  plasma  may  vary  considerably 
under  different  circumstances  even  in  the  same  species  of  animal.  In 
animals  in  most  cases  considerably  more  plasma  is  found,  sometimes  two- 
thirds  of  the  weight  of  the  blood.3  For  human  blood  Arronet  has  found 
478.S  p.  m.  blood-corpuscles  and  521.2  p.  m.  serum  (in  defibrinated  blood) 
as  an  average  of  nine  determinations.  Schneider  *  found  349.6  and  650.4 
p.  m.  respectively  in  women. 

The  sugar  occurs,  it  seems,  only  in  the  serum  and  not  with  the  blood- 
corpuscles.  The  same  is  true,  according  to  Abderhalden,  for  the  limer 
fat,  and  perhaps  also  the  fatty  acids.  The  small  traces  of  bile-acids  found 
in  normal  blood  is,  according  to  Croftan,5  contained  in  the  leucocytes. 
The  division  of  the  alkalies  between  the  blood-corpuscles  and  the  plasma 
is  different,  as  the  blood-corpuscles  from  the  pig,  horse,  and  rabbit  contain 
no  soda,  those  from  human  blood  are  richer  in  potassium,  and  the  corpuscles 
from  ox-,  sheep-,  goat-,  dog-,  and  cat-blood  are  considerably  richer  in 
sodium  than  potassium.  Chlorine  exists  in  all  blood  to  a  greater  extent 
in  the  serum  than  in  the  blood-corpuscles.  Iodine  is  only  found  in  the 
serum,  while  iron  occurs  chiefly  in  the  form-elements,  especially  in  the 
erythrocytes.  As  the  nucleoproteids  contain  iron,  some  iron  always  occurs 
in  the  erythrocytes  and  traces  of  iron  are  also  found  in  the  serum.  This 
amount  under  normal  conditions  is'  very  small,  while  in  disease  the  relation 
between  haemoglobin-iron  and  other  blood-iron  does  not  seem  to  change 
very  much.  There  is  also  found  in  the  blood  manganese  and  traces  of  lith- 
ium, copper,  lead,  silver,  and  in  menstrual  blood  arsenic  lias  also  been  noted. 
The  blood  as  a  whole  contains  in  ordinary  cases  770-820  p.  m.  water,  with 
180-230  p.  m.  solids;  of  these  173-220  p.  m.  are  organic  and  6-10  p.  m. 
inorganic.  The  organic  consists,  deducting  6-12  p.  m.  extractive  bodies, 
of  proteids  and  haemoglobin.  The  amount  of  this  last-mentioned  body  in 
hui nan  blood  is  about  130-150  p.  m.     In  the  dog,  cat,  pig,  and  horse  the 

1  Zeitschr.  f.  physiol.  Chem.,  23  and  25. 

:  Cited  and  in  part  recalculated  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem., 
4.  Aufl.,  345. 

3  See  Sacharjin  in  Hoppe-Seyler's  Physiol.  Chem.,  447;  Otto,  Pfliiger's  Arch.,  35; 
Bunge,  1.  c. ;  L.  and  M.  Bleibtreu,  Pfliiger's  Arch.,  51. 

4  Arronet,  Maly's  Jahresber.,  17;   Schneider,  Centralbl.  f.  Physiol.,  5,  362. 

5  Pfliiger's  Arch.,  90. 


202  THE  BLOOD. 

quantity  of  haemoglobin  is  about  the  same,  and  lower  in  the  blood  from  the 
ox,  bull,  sheep,  goat,  and  rabbit  (Abderhalden). 

The  amount  of  sugar  in  the  blood  is  on  an  average  1-1.5  p.  m.  It 
seems  to  be  independent  of  the  composition  of  the  food,  but  feeding 
with  large  amounts  of  sugar  or  dextrin  causes  a  considerable  increase  in 
the  sugar  of  the  blood,  as  observed  by  Bleile.  When  the  quantity  of 
sugar  amounts  to  more  than  3  p.  m.,  then,  according  to  Cl.  Bernard,1 
sugar  appears  in  the  urine,  and  a  glycosuria  appears.  An  increase  in  the 
quantity  of  sugar  takes  place,  as  first  observed  by  Bernard  and  lately 
substantiated  by  Fr.  Schenck,  after  removal  of  blood.  According  to 
Hexriques  2  this  increase  of  the  reducing  power,  at  least  in  dogs,  is  not 
due  to  sugar  but  chiefly  to  jecorin,  which  substance  is  the  cause  of  more  of 
the  reduction  in  normal  blood  than  the  sugar.  It  is  difficult  to  judge  of  the 
value  of  many  statements  as  to  the  amount  of  sugar  and  reducing  power  of 
the  blood,  because  the  experimentors  generally  have  not  considered  the 
presence  of  a  certain  quantity  of  jecorin  or  conjugated  glucuronic  acids  or 
they  were  unable  to  do  so. 

The  quantity  of  urea,  which,  according  to  Schondorff,  is  equally  divided 
between  the  blood-corpuscles  and  the  plasma,  is  greater  on  taking  food  than 
in  starvation  (Grehant  and  Quinquaud,  Schondorff)  and  varies  between 
0.2  and  1.5  p.  m.  In  dogs  Schondorff  found  in  starvation  a  minimum 
of  0.348  p.  m.  and  a  maximum  of  1.529  p.  m.  at  the  point  of  highest 
urea  formation.  Gottlieb  obtained  much  lower  results  by  another  direct 
method,  namely,  in  starvation  0.1  to  0.2,  and  after  meat  feeding  0.28-0.56, 
p.  m.  In  man  v.  Jaksch  3  found  0.5-0.6  p.  m.  urea  in  normal  blood.  The 
quantity  of  urea  is  somewhat  increased  in  fever  and  in  general  in  augmented 
proteid  metabolism  and  the  increased  urea  formation  depending  thereon. 
A  more  important  increase  in  the  quantity  of  urea  in  the  blood  occurs  in  a 
retarded  elimination  of  urea,  as  in  cholera,  also  in  cholera  infantum  and 
in  infections  of  the  kidneys  and  the  urinary  passages.  After  ligaturing 
the  ureters  or  after  extirpation  of  the  kidneys  of  animals  an  accumulation 
of  urea  takes  place  in  the  blood. 

The  blood  also  contains  traces  of  ammonia.  According  to  Horodynski, 
Salaskin,  and  Zaleski,4  who  worked  with  the  improved  Nencki  and 
Zalesei  method,  the  quantity  in  arterial  dog-blood  was  0.41  milligram  in 
100  grams  of  blood.      The  blood  of  the  portal  vein  contains  considerably 

1  Bleile,  Du  Bois-Reymond  's  Arch. ,  1879 ;  Bernard,  Lecons  sur  le  diabete.  Paris,  1877. 

2  Schenck,  Pfliiger's  Arch.,  57;  Henriques,  Zeitschr.  f.  physiol.  Chem.,  28.  See  also 
Kolisch  and  Stejskal,  Wien.  klin.  Wochenschr. ,  1898. 

3  Grehant  et  Quinquaud,  Journ.  de  l'anatomie  et  de  la  physiol.,  20,  and  Compt. 
rend.,  98;  Schondorff,  Pfliiger's  Arch.,  54  and  63;  Gottlieb,  Arch.  f.  expt.  Path.  u. 
Pharm.,  42;  v.  Jaksch.,  Leyden-Fcstschr. ,  I,  1901. 

*  Zeitschr.  f.  physiol.  Chem.,  35,  which  also  gives  the  older  literature. 


BLOOD  IN  DIFFERENT   VASCULAR  REGIONS.  203 

more  than  the  blond  of  the  arteries,  namely  3-4.5  times  richer;   this    is 
disputed  by  Biedl  and  Winterberg,1  however. 

The  blood  from  healthy  persons  contains  on  an  average  0.90  milligram 
per  100  c.  c-  according  to  Winterberg."  The  quantity  of  uric  acid  may 
be  0.1  p.  m.  in  bird's  blood  (v.  Schroder4).  Uric  acid  has  not  been 
detected  with  positivenesa  in  human  blood  under  normal  conditions,  while 
it  has  been  found  in  the  blood  in  gout,  croupous  pneumonia,  and  certain 
other  diseased  conditions.  Lactic  acid  was  first  found  in  human  blood  by 
Salomon  and  then  by  (Iaglio,  Berlinerblau,  and  Irisawa.  The  quan- 
tity of  lactic  acid  may  vary  considerably.  Berlinerblau  found  0.71  p.  m. 
as  maximum.  Saito  and  Katsutama  5  found  on  an  average  0.269  p.  m. 
in  hen's  blood,  and  after  carbon-monoxide  poisoning  the  quantity  increased 
to  1.227  p.  m. 

The  Composition  of  the  Blood  in  Different  Vascular  Regions  and  under 

Different  Conditions. 

Arterial  and  Venous  Blood.  The  most  striking  difference  between 
these  two  kinds  of  blood  is  the  variation  in  color  caused  by  their  containing 
different  amounts  of  gas  and  different  amounts  of  oxyhemoglobin  and 
haemoglobin.  The  arterial  blood  is  light  red;  the  venous  blood  is  dark  red, 
dichroitic,  greenish  by  transmitted  light  through  thin  layers.  The  arterial 
coagulates  more  quickly  than  the  venous  blood.  The  latter,  on  account  of 
the  transudation  which  takes  place  in  the  capillaries,  is  somewhat  poorer  in 
water  but  richer  in  blood-corpuscles  and  haemoglobin  than  the  arterial 
blood;  but  this  is  denied  by  modern  investigators.  According  to  Kruger* 
and  his  pupils  the  quantity  of  dry  residue  and  haemoglobin  in  blood  from 
the  carotid  artery  and  from  the  jugular  vein  (in  cats)  is  the  same.  Roh- 
m.v.vx  and  MrusAM7  could  not  detect  any  difference  in  the  quantity  of 
fat  in  arterial  and  venous  blood. 

Blood  from  the  Portal  Vein  and  tin  Hepatic  Vein.  In  consequence  of 
the  small  quantities  of  bile  and  lymph  found  relatively  to  the  large  quantity 
of  blood  circulating  through  the  liver  in  a  given  time  we  can  hardly  expect 
to  detect    by  chemical  analysis  a    positive  difference  in  the  composition 

1  r Auger's  Arch.,  88. 

:  Ascoli,  Ibid.,  87,  has  suggested  a  method  for  the  quantitative  estimation  of  the 
extractive  nitrogen  in  Mood. 

3  Wien.  klin.  Wochenschr.,  1897,  and  Zeitschr.  f.  klin.  Med..  35. 

••Ludwig'a  Festschrift,  1S87. 

'Irisawa,  Zeitschr.  f.  physiol.  Chem.,  17,  which  also  gives  the  elder  literature;  SaitO 
and  Katsuvama,  ibid.,  32. 

8  Zeitschr.  f.  Biologie,  26.  This  also  gives  the  literature  on  the  composition  of  the 
blood  in  different  vascular  regions. 

'Pfluger's  Archiv,  46. 


204  THE  BLOOD. 

between  the  blood  of  the  portal  and  hepatic  veins.  The  statements  in 
regard  to  such  a  difference  are  in  fact  contradictory.  For  example,  Dros- 
doff  has  found  more  haemoglobin  in  the  hepatic  than  in  the  portal  vein,  while 
Otto  found  less.  Kruger  finds  that  the  quantity  of  haemoglobin,  as 
well  as  the  solids,  in  the  blood  from  the  vessels  passing  to  and  from  the 
liver  is  different,  but  a  constant  relationship  cannot  be  determined.  The 
disputed  question  as  to  the  varying  quantities  of  sugar  in  the  portal  and 
hepatic  veins  will  be  discussed  in  a  following  chapter  (see  Chapter  VIII, 
on  the  formation  of  sugar  in  the  liver).  After  a  meal  rich  in  carbohy- 
drates the  blood  of  the  portal  vein  not  only  becomes  richer  in  dextrose, 
but  may  contain  also  dextrin  and  other  carbohydrates  (v.  Merixg,  Otto  j). 
The  amount  of  urea  in  the  blood  from  the  hepatic  vein  is  greater  than 
in  other  blood  (Grehaxt  and  Quixquaud  2) .  In  regard  to  the  quantity 
of  ammonia,  see  page  202. 

Blood  of  the  Splenic  Vein  is  decidedly  richer  in  leucocytes  than  the 
blood  from  the  splenic  artery .  The  red  blood-corpuscles  of  the  blood  from 
the  splenic  vein  are  smaller  than  the  ordinary,  less  flattened,  and  show  a 
greater  resistance  to  water.  The  blood  from  the  splenic  vein  is  also  claimed 
to  be  richer  in  water,  fibrin,  and  proteid  than  the  ordinary  venous  blood. 
According  to  v.  Middexdorff,  it  is  richer  in  haemoglobin  than  arterial 
blood.  Kruger  3  and  his  pupils  have  found  that  the  blood  from  the  vena 
lienalis  is  generally  richer  in  haemoglobin  and  solids  than  arterial  blood; 
still  the  contrary  is  often  found.  The  blood  from  the  splenic  vein  coagu- 
lates slowly. 

The  Blood  from  the  Veins  of  the  Glands.  The  blood  circulates  with 
greater  rapidity  through  a  gland  during  activity  (secretion)  than  when  at 
rest,  and  the  outflowing  venous  blood  has  therefore  during  activity  a  lighter 
red  color  and  a  greater  amount  of  oxygen.  Because  of  the  secretion  the 
venous  blood  also  becomes  somewhat  poorer  in  water  and  richer  in  solids. 

The  blood  from  the  Muscular  Veins  shows  an  opposite  behavior,  for 
during  activity  it  is  darker  and  more  venous  in  its  properties  because  of  the 
increased  absorption  of  oxygen  by  the  muscles  and  still  greater  production 
of  carbon  dioxide  than  when  at  rest. 

Miiislrii.nl  Blood  has,  according  to  an  old  statement,  not  the  power  of 
coagulating.  This  statement  is  nevertheless  false,  and  the  apparent  un- 
coagi  liability  depends  in  part  on  the  womb  and  the  vagina  retaining  the 
blood-clot,  so  that  only  fluid  cruor  is  at  times  eliminated,  and  in  part  on  a 
contamination  with  vaginal  mucus,  which  disturbs  the  coagulation.     Men- 


1  DrosdofF,  Zeitechr.  f.  phyeiol.  Chem.,  1;  Otto,  Maly's  Jahresber.,  17;   v.  Mering, 
Du  Bois-Reymond's  Arch.,  1877,  214. 
2L.  c. 
'v.  Middendorff,  Centralbl.  f.  Physiol.,  2,  753;  Kruger,  L  c. 


THE  BLOOD  AT  DIFFERENT  PERIODS  OF  LIFE.  205 

strual  blood,  according  to  Gautier  and  Bourcet,  contains  arsenic  and  is 
also  richer  in  iodine  than  other  blood  (see  blood-serum,  page  157). 

77/c  Blood  of  tin  Tiro  Sexes.  Woman's  blood  coagulates  somewhat  more 
quickly,  has  a  lower  specific  gravity,  a  greater  amount  of  water,  and  a 
smaller  quantity  of  solids  than  the  blood  of  man.  The  amount  of  blood- 
corpuscles  and  hemoglobin  is  somewhat  smaller  in  woman's  blood.  The 
amount  of  haemoglobin  is  146  p.m.  for  man's  blood  and  133  p.m.  for 
woman's. 

During  pregnancy  Nasse  has  observed  a  decrease  in 'the  specific  gravity, 
with  an  increase  in  the  amount  of  water  until  the  end  of  the  eighth  month. 
From  then  the  specific  gravity  increases,  and  at  delivery  it  is  normal  again. 
The  amount  of  fibrin  is  somewhat  increased  (Becquerel  and  Rodier, 
Nasse).  The  number  of  blood-corpuscles  seems  to  decrease.  In  regard  to 
the  amount  of  haemoglobin  the  statements  are  somewhat  contradictory. 
Cohxsteix  found  the  number  of  red  corpuscles  diminished  in  the  blood 
of  pregnant  sheep  as  compared  to  non-pregnant,  but  the  red  corpuscles 
were  larger  and  the  quantity  of  haemoglobin  in  the  blood  was  greater  in  the 
first  case.  Mollenbebg  '  found  in  most  cases  an  increase  in  the  amount 
of  haemoglobin  in  pregnancy  in  the  last  months. 

The  Blood  at  Different  Periods  of  Life.  Foetal  and  infant  blood  is  richer 
in  erythrocytes  and  haemoglobin  than  the  blood  of  the  mother.  The 
highest  percentage  of  haemoglobin  in  the  blood  has  been  observed  by 
several  investigators  such  as  Cohnstein  and  Zuntz,  Otto,  YStxterxitz, 
Abderhaldex,  Schwixge,  and  others  immediately  or  very  soon  after 
birth  or  at  least  within  the  first  days.  In  man  two  or  three  days  after 
birth  the  haemoglobin  reaches  a  maximum  (200-210  p.  m.),  which  is  greater 
than  at  any  other  period  of  life.  This  is  the  cause  of  the  great  abundance 
of  solids  in  the  blood  of  new-born  infants  as  observed  by  several  inves- 
tigators. The  quantity  of  haemoglobin  and  blood-corpuscles  sinks  gradu- 
ally from  this  first  maximum  to  a  minimum  of  about  110  p.  m.  haemoglobin, 
which  minimum  appears  in  human  beings  between  the  fourth  and  eighth 
3-ears.  The  quantity  of  haemoglobin  then  increases  again  until  about  the 
twentieth  year,  when  a  second  maximum  of  137-150  p.  m.  is  reached.  The 
haemoglobin  remains  at  this  point  only  towards  the  forty-fifth  year,  and 
then  gradually  and  slowly  decreases  (Leichtexsterx,  Otto2).  According 
to  older  statements,  the  blood  at  old  age  is  poorer  in  blood-corpuscles  and 
proteid  bodies  but  richer  in  water  and  salts. 

1  Xasse,  Maly's  Jahresber.,  7;  Becquerel  and  Rodier,  Traite  de  chim.  pat  hoi. 
Paris,  1854;  Cohnstein,  Pfliiger's  Arch.,  34,  233;  Mollenberg,  Maly's  Jahresber.,  31, 
185. 

2  Cohnstein  and  Zuntz,  Pfliiger's  Arch.,  34;  Winternitz,  Zeitschr.  f.  physiol.  Chem., 
22;  Leichtenstern,  t'ntersuch.  iiber  den  Hamoglobingehalt  des  Blutes,  etc.  Leipzig, 
1878; — Otto,  Maly's  Jahresber.,  15  and  1";  Abderhalden,  Zeitschr.  f.  physiol.  Chem., 
34j  Schwinge,  Pfliiger's  Arch.,  73  (literature). 


206  THE  BLOOD. 

The  Influence  o]  Food  on  the  Blood.  In  complete  starvation  no  decrease 
in  the  amount  of  solid  blood  constituents  is  found  to  take  place  (Panum 
and  others).  The  amount  of  haemoglobin  is  increased  a  little,  at  least  in 
the  early  period  (Subbotin,  Otto,  Hermann  and  Groll,  Luciani  and 
Bufalini),  and  also  tne  number  of  red  blood-corpuscles  increases  (Worm. 
Muller,  Buntzen  *),  which  probably  depends  on  the  fact  that  the  blood- 
corpuscles  are  not  so  quickly  transformed  as  the  serum  and  partly  because 
of  a  greater  concentration  due  to  loss  of  water.  In  rabbits  and  to  a  less 
extent  in  dogs,  Popel  found  that  complete  abstinence  had  a  tendency 
to  increase  the  specific  gravity  of  the  blood.  The  amount  of  fat  in  the- 
blood  may  be  somewhat  increased  in  starvation  because  the  fat  is  taken 
up  from  the  fat  deposits  and  carried  to  the  various  organs  by  the  blood 
(N.  Schulz,  Daddi  2) . 

After  a  rich  meal  the  relative  number  of  blood-corpuscles,  after  secretion 
of  digestive  juices  or  absorption  of  nutritive  liquids,  may  be  increased  or 
diminished  (Buntzen,  Leichtenstern)  .  The  number  of  white  blood- 
corpuscles  may  be  considerably  increased  after  a  diet  rich  in  proteids. 
After  a  diet  rich  in  fat  the  plasma  becomes,  even  after  a  short  time,  more 
or  less  milky-white,  like  an  emulsion.  The  composition  of  the  food  acts 
essentially  on  the  amount  of  haemoglobin  in  'the  blood.  The  blood  of 
herbivora  is  generally  poorer  in  haemoglobin  than  that  from  carnivora, 
and  Subbotin  has  observed  in  dogs  after  a  partial  feeding  with  food  rich 
in  carbohydrates  that  the  amount  of  haemoglobin  sank  from  the  physiological 
average  of  137.5  p.  m.  to  103.2-93.7  p.  m.  Tsuboi  3  has  also  shown  in 
experiments  on  rabbits  and  dogs  that  with  an  insufficient  diet  of  bread 
and  potatoes,  where  the  body  gave  up  proteid  and  contained  relatively 
considerable  carbohydrate,  the  amount  of  haemoglobin  decreased  and 
the  blood  became  richer  in  water.  According  to  Leichtenstern  a 
gradual  increase  in  the  amount  of  haemoglobin  is  found  to  take  place  in 
the  blood  of  human  beings  on  enriching  the  food,  and  according  to  the 
same  investigator  the  blood  of  lean  persons  is  generally  somewhat  richer  in 
haemoglobin  than  blood  from  fat  ones  of  the  same  age.  The  addition  of 
iron  salts  to  the  food  greatly  influences  the  number  of  blood-corpuscles  and 
especially  the  amount  of  haemoglobin  they  contain.  The  action  of  the  iron 
salts  is  obscure.4    There  does  not  seem  to  be  any  doubt  that  not  only  is 

1  Panum,  Virchow's  Arch.,  29;  Subbotin,  Zeitschr.  f.  Biologie,  7;  Otto,  1.  c. ;  Worm. 
Muller,  Transfusion  und  Plethora.  Christiania,  1875; — Buntzen,  see  Maly's  Jahresber., 
9;  Hermann  and  Groll,  Pfliiger's  Arch.,  43;  Luciani  and  Bufalini,  Maly's  Jahresber. ,  12- 

2  Popel,  Arch,  des  scienc.  biol.  de  St.  Petersbourg,  4,  354;  Schulz,  Pfliiger's  Arch., 
65;   Daddi,  Maly's  Jahresber.,  30. 

3  Subbotin,  1.  c. ;  Tsuboi,  Zeitschr.  f.  Biologie,  44. 

4  See  Bunge,  Zeitschr.  f.  physiol.  Chem.,  9;  Iliiusermann,  ibid.,  23,  where  the  works 
of  Woltering,  Gaule,  Hall,  Hochhaus,  and  Quincke  are  cited.  The  same  work  con- 
tains a  table  of  the  quantity  of  iron  in  various  foods;  Kunkel,  Pfliiger's  Arch.,  61; 
Macullum,  Journal  of  Physiol.,  16;   Abderhalden,  Zeitschr.  f.  Biologie.  39 


NUMBER  OF   BLOOD  CORPUSCLES.  207 

the  iron  contained  in  the  food  in  the  form  of  organic  compounds  active, 
but  also  iron  salts  and  therapeutic  iron.  According  to  BtTNGE  and  his 
pupils  the  iron  preparations  only  act  indirectly.  They  may  combine  with 
the  sulphuretted  hydrogen  of  the  intestinal  canal  and  thereby  prevent  the 
iron  associated  in  the  food  as  assimilable,  protein  compounds  from  being 
eliminated  as  iron  sulphide  (Bunoe),  or  they  may  perhaps  act  as  irritants 
upon  the  blood  forming  organs  (Abderhalden). 

An  increase  in  the  number  of  red  corpuscles,  a  true  "plethora  POLT- 
cyth.emia,"  takes  place  after  transfusion  of  blood  of  the  same  species  of 
animal.  According  to  the  observations  of  Panum  and  Worm  Mi  ller,1 
the  blood-liquid  is  quickly  eliminated  and  transformed  in  this  case, — the 
water  being  eliminated  principally  by  the  kidneys  and  the  proteid  burned 
into  urea,  etc., — while  the  blood-corpuscles  are  preserved  longer  and  cause 
a  "  polycythemia."  A  relative  increase  in  the  number  of  red  corpuscles 
is  found  after  abundant  transudation  from  the  blood,  as  in  cholera  and 
heart-failure  with  considerable  congestion.  An  increase  in  the  number 
of  red  blood-corpuscles  has  also  been  observed  under  the  influence  of 
diminished  pressure  or  in  high  altitudes.  Viault  first  called  attention  to 
the  fact  that  the  number  of  red  corpuscles  was  very  great  in  the  blood  of 
man  and  animals  living  in  high  regions.  According  to  him  the  llama  has 
about  16  million  blood-corpuscles  per  cubic  millimeter.  By  observations 
on  himself  and  others,  as  well  as  on  animals,  Viault  found  the  first  effect 
of  sojourning  in  high  localities  was  a  very  considerable  increase  in  the 
number  of  red  corpuscles,  in  his  own  case  5-8  millions.  A  similar  increase 
of  the  red  blood-corpuscles,  as  also  an  increase  in  the  quantity  of  haemo- 
globin under  the  influence  of  diminished  pressure,  has  been  observed  by 
many  other  investigators  in  human  beings  as  well  as  in  animals.  Investi- 
gators are  not  united  as  to  how  this  increase  is  brought  about.  The  increase 
in  the  blood-corpuscles  is  not  absolute  but  is  only  relative,  and  it  is  con- 
sidered by  several  observers  that  there  is  neither  a  new  formation  (Viault 
and  others)  nor  a  diminished  destruction  of  the  blood-corpuscles  (Fick).  A 
relative  increase  may  be  brought  about  in  different  ways.  For  example, 
another  division  of  the  blood-corpuscles  in  the  vascular  system  has  been 
considered,  where,  in  the  capillaries,  from  which  region  the  blood  has  been 
examined  more  often,  the  blood-corpuscles  accumulate  (Zuntz).  It  is  also 
considered  that  a  concentration  of  the  blood  takes  place  by  increased  evapo- 
ration (Grawitz),  and  finally  an  increase  in  the  blood-corpuscles  has  also 
been  explained  by  a  contraction  of  the  vascular  system  with  the  pressing  out 
of  plasma  (Bunge,  Abderhalden  2). 

A  decrease  in  the  number  of  red  corpuscles  occurs  in  anaemia  from  differ- 

1  Panum,  Virchow's  Arch.,  25);    Worm  Muller,  1.  c. 

2  The  literature  on  this  subject  may  be  found  in  Abderhalden,  Zeitschr.  f.  Biologie, 
43;   van  Voornveld,  Pfluger's  Arch.,  92. 


208  THE  BLOOD. 

ent  causes.  Even-  excessive  hemorrhage  causes  an  acute  anaemia,  or,  more 
correctly,  oligaemia.  Even  during  the  hemorrhage  the  remaining  blood 
becomes  by  diminished  secretion  and  excretion,  as  also  by  an  abundant 
absorption  of  parenchymous  fluid,  richer  in  water,  somewhat  poorer  in  pro- 
teids,  and  strikingly  poorer  in  reel  blood-corpuscles.  The  oligsemia  passes 
soon  into  a  hydrsemia.  The  amount  of  proteid  then  gradually  increases 
again ;  but  the  reformation  of  the  red  blood-corpuscles  is  slower,  and  after 
the  hydrsemia  follows  also  an  oligocythaemia.  After  a  little  time  the 
number  of  blood-corpuscles  rises  to  normal;  but  the  reformation  of  haemo- 
globin does  not  keep  pace  with  the  reformation  of  the  corpuscles,  and  a 
chlorotic  condition  may  appear.  A  considerable  decrease  in  the  number 
of  red  corpuscles  occurs  also  in  chronic  anaemia  and  chlorosis;  still  in  such 
cases  an  essential  decrease  in  the  amount  of  haemoglobin  occurs  without  an 
essential  decrease  in  the  number  of  blood-corpuscles.  The  decrease  in  the 
amount  of  haemoglobin  is  more  characteristic  of  chlorosis  than  a  decrease 
in  the  number  of  red  corpuscles.  The  statements  on  the  changes  in  the 
blood  in  anaemia  and  chlorosis  differ  very  considerably,  and  in  this  con- 
nection attention  must  be  called  to  the  findings  of  Lorrain  Smith  (based  on 
his  estimation  of  the  oxygen  capacity  and  the  blood  volume)  that  for  chlorosis 
an  absolute  diminution  of  the  amount  of  haemoglobin  is  not  essential,  while 
the  total  quantity  of  haemoglobin  may  be  normal,  but  only  a  relative 
diminution  occurs,  due  to  a  pronounced  increase  of  the  blood-plasma  and 
of  the  total  quantity  of  blood.1 

A  very  considerable  decrease  in  the  number  of  red  corpuscles  (300,000- 
400,000  in  1  c.  mm.)  and  diminution  in  the  amount  of  haemoglobin  (£-rV) 
occurs  in  pernicious  anaemia  (Hayem,  Laache,  and  others).  On  the 
contrary,  the  individual  red  corpuscles  are  larger  and  richer  in  haemoglobin 
than  they  ordinarily  are,  and  the  number  stands  in  an  inverse  relationship 
to  the  amount  of  haemoglobin  (Hayem).  Besides  this  the  red  corpuscles 
often,  but  not  always,  show  in  pernicious  anaemia  remarkable  and  extraor- 
dinary irregularities  of  form  and  size,  which  Quincke  2  has  termed  poikilo- 
cyiosis. 

The  number  of  leucocytes  may,  as  stated  above,  be  increased  under 
physiological  conditions  as  well  as  after  a  meal  rich  in  proteid.  Under 
pathological  conditions  a  high  leucocytosis  may  occur,  and  this  is  especially 
found  in  leucaemia,  which  is  characterized  by  a  very  great  abundance  of 
leucocytes  in  the  blood.  The  number  of  leucocytes  is  markedly  increased 
in  this  disease,  and  indeed,  not  only  absolutely,  but  also  in  relation  to  the 

1  Trans.  Path.  Soc.  London,  51,  1900.  Complete  analyses  of  chlorotic  blood  may- 
be found  in  Erben,  Zeitschr.  f.  klin.  Med.,  47. 

2  Laache,  "Die  Anamie"  (Christiania,  1883),  which  also  contains  the  literature] 
Quincke,  Deutsch.  Arch.  f.  klin.  Med.,  20  and  25.  A  complete  chemical  analysis  of 
the  blood  has  been  made  by  Erben,  Zeitschr.  f.  klin.  Med.,  40. 


BLOOD  IN  DISEASE.  209 

number  of  red  blood-corpuscles,  which  arc  increased  to  a  considerable  extent 
in  leucaemia.  Leucsemic  blood  lias  a  lowerspecific  gravity  than  the  ordinary 
blood  (1035-1040),  and  a  paler  color,  as  if  it  was  mixed  with  pus.  The  reaction 
is  alkaline, and  after  death  it  is  often  acid,  probably  due  to  a  decomposition 
of  lecithin,  which  is  often  considerably  increased  in  leucaemia.  Volatile 
fatty  acids,  lactic  acid,  glycerophosphoric  acid,  large  amounts  of  xanthine 
bodies  and  so-called  Charcot's  crystals  (see  semen,  page  420)  have  also 
been  found  in  leucsemic  blood.  The  peptone  (proteose)  which  is  found  in 
the  leucsemic  blood  after  death,  and  which  does  not  exist  in  the  fresh  blood, 
is,  according  to  Erben,  a  digestive  product  which  is  produced  by  a  tryptic 
enzyme  as  well  as  traces  of  a  peptic  enzyme,  which  originate  from  the  leuco- 
cytes These  enzymes,  according  to  Erben,  do  not  occur  in  normal  blood, 
or  are  so  firmly  combined  therein  that  on  the  death  of  the  cells  they  are 
not  set  free,  or  at  least  their  action  does  not  become  evident.1 

There  are  only  a  few  complete  analyses  of  the  chemical  composition 
of  blood  in  disease,  still  a  great  number  of  investigations  have  been  made 
on  this  subject.  But  as  we  have  only  a  fewr  analyses  of  the  blood  of  healthy 
individuals,  and  as  the  possible  variation  under  physiological  conditions  is 
little  known,  it  is  difficult  to  draw  any  positive  conclusions  from  the  analyses 
of  pathological  blood.  Unfortunately  on  account  of  the  large  number  of 
contradictory  statements  of  the  composition  of  the  blood  of  diseased  human 
beings  it  is  impossible  to  give  a  brief  summary  of  the  results,  still  the  changes 
in  the  blood  in  disease  must  be  of  the  greatest  value. 

The  quantity  of  blood  is  indeed  somewhat  variable  in  different  species 
of  animals  and  in  different  conditions  of  the  body;  in  general  we  consider 
the  entire  quantity  of  blood  in  adults  as  about  tV-!1*  °f  the  weigh.1  of  the 
body,  and  in  new-born  infants  about  -fa.  Haldaxe  and  LoRRAIN  Smith,2 
who  have  determined  the  quantity  of  blood  by  a  new  method,  find  in  fourteen 
persons  that  it  varies  between  T^  and  -fa  of  the  weight  of  the  body.  Fat 
individuals  are  relatively  poorer  in  blood  than  lean  ones.  During  inanition 
the  quantity  of  blood  decreases  less  quickly  than  the  weight  of  the  body 
(Pantjm8),  and  it  may  therefore  be  also  proportionally  greater  in  starving 
individuals  than  in  well-fed  ones. 

By  careful  bleeding  the  quantity  of  blood  may  be  considerably  dimin- 
ished without  any  dangerous  symptoms.  The  loss  of  blood  to  one  fourth 
of  the  normal  quantity  has  as  a  sequence  no  durable  sinking  of  the  blood- 
pressure  in  the  arteries,  because  the  smaller  arteries  accommodate  them- 
selves to  the  small  quantities  of  blood  by  contracting  (Worm  Muller  4).  A 
loss  of  blood  to  one  third  of  the  quantity  reduces  the  blood-pressure  con* 

. 1 — v 

1  Erben,  Zeitschr.  f.  Heilkunde,  24. 

2  Journ.  of  Physiol.,  25. 
3Yirehow's  Arch.,  20. 

*  Transfusion  und  Plethora,  Christiania,  1875. 


210  THE  BLOOD. 

siderably,  and  a  loss  of  one  half  of  the  blood  in  adults  is  dangerous  to  life. 
The  more  rapid  the  bleeding  the  more  dangerous  it  is.  New-born  infants 
are  very  sensitive  to  loss  of  blood,  and  likewise  fat,  old,  and  weak  persons 
cannot  stand  much  loss  of  blood.  Women  can  stand  loss  of  blood  better 
than  men. 

The  quantity  of  blood  may  be  considerably  increased  by  the  injection  of 
blood  from  the  same  species  of  animal  (Panum,  Laxdois,  Worm  Muller, 
Poxfick).  According  to  Worm  Muller  the  normal  quantity  of  blood  may 
indeed  be  increased  to  83  per  cent  without  producing  any  abnormal  condi- 
tions or  lasting  high  blood-pressure.  An  increase  of  the  quantity  of  blood 
to  150  per  cent  may,  with  a  considerable  variation  in  the  blood-pressure, 
be  directly  dangerous  to  life  (Worm  Muller).  If  the  quantity  of  blood 
of  an  animal  is  increased  by  transfusion  with  blood  of  the  same  kind  of 
animal ,  an  abundant  formation  of  lymph  takes  place.  The  water  in  excess 
is  eliminated  by  the  urine;  and  as  the  proteid  of  the  blood-serum  is  quickly 
decomposed,  while  the  red  blood-corpuscles  are  destroyed  much  more 
slowly  (Tschir je w ,  Forster  ,  Paxum  ,  Worm  Muller  *) ,  a  polycythemia 
is  gradually  produced. 

The  quantity  of  blood  in  the  different  organs  depends  essentially  on  the 
activity  of  the  same.  During  work  the  exchange  of  material  in  an  organ 
is  more  pronounced  than  when  at  rest,  and  the  increased  metabolism  is  con- 
nected with  a  more  abundant  flow  of  blood.  Although  the  total  quantity  of 
blood  in  the  body  remains  constant  the  distribution  of  the  blood  in  the 
various  organs  may  be  different  at  different  times.  As  a  rule  the  quantity 
of  blood  in  an  organ  can  be  an  approximate  measure  of  the  more  or  less 
active  metabolism  going  on  in  the  same,  and  from  this  point  of  view  the 
distribution  of  the  blood  in  the  different  organs  and  groups  of  organs  is  of 
interest.  According  to  Raxke,2  to  whom  we  are  especially  indebted  for 
our  knowledge  of  the  relationship  of  the  activity  of  the  organs  to  the 
quantity  of  blood  contained  therein,  of  the  total  quantity  of  blood  (in  the 
rabbit)  about  one  fourth  comes  to  the  muscles  in  rest,  one  fourth  to  the 
heart  and  the  large  blood-vessels,  one  fourth  to  the  liver,  and  one  fourth  to 
the  other  organs. 


1  Panum,  Xord.  med.  Ark.,  7;  Virchow's  Arch.,  03;  Landois,  Centralbl.  f.  d.  med. 
Wi    enscb  d  "Die  Transfusion  des  Blutes,"  Leipzig,  1875;    "Worm  Muller, 

>    fusion  und  Plethora";   Ponfick,  Virchow's  Arch.,  <>2;   Tschirjew,  Arheiten  aus 
der  Physiol.  Anstalt  zu  Leipzig,  1874,  292;   Forster,  Zeitschr.  f.  Biologie,  11;   Panum, 
\rcli.,  2;). 

2  Die  Blutvertheilung  und  der  Thatigkeitswecbsel  der  Organe,  Leipzig,  1871. 


CHAPTER  MI. 

CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

I.  Chyle  and  Lymph. 

The  lymph  is  the  mediator  in  the  exchange  of  constituents  between  the 
blood  and  tissues.  The  bodies  necessary  for  the  nutrition  of  the  tissues 
pass  from  the  blood  into  the  lymph,  and  the  tissues  deliver  water,  salts,  and 
products  of  metabolism  to  the  lymph.  The  lymph,  therefore,  originates 
partly  from  the  blood  and  partly  from  the  tissues.  From  a  purely  theoret- 
ical standpoint  one  can,  according  to  Heidexiiaix,  differentiate  between 
blood-lymph  and  tissue-lymph  according  to  origin.  It  is  impossible  at  the 
present  time  to  completely  separate  that  which  comes  from  the  one  or  the 
other  source.  Chemically  the  lymph  is  the  same  as  plasma  and  contains,  at 
least  to  a  great  extent,  the  same  bodies.  The  observation  of  Asher  and 
Bakrera,1  that  the  lymph  contains  poisonous  metabolic  products,  does 
not  contradict  such  an  assumption,  as  no  doubt  these  products  are  trans- 
ferred to  the  blood  with  the  lymph.  Although  the  blood  does  not  show  the 
same  poisonous  action  as  the  lymph,  still  this  can  be  explained  by  the 
great  dilution  these  bodies  undergo  in  the  blood,  and  the  difference  between 
blood-plasma  and  lymph  is  no  doubt  of  a  quantitative  nature.  This  differ- 
ence consists  chiefly  in  that  the  lymph  is  poorer  in  proteids.  No  essen- 
tial chemical  difference  has  been  found  between  the  lymph  and  the  chyle 
of  starving  animals.  After  fatty  food  the  chyle  differs  from  the  lymph  in 
its  wealth  of  minutely  divided  fat-globules  which  give  it  a  milky  appear- 
ance; hence  the  old  name  "lacteal  fluid." 

Chyle  and  lymph,  like  the  plasma,  contain  seralbumin,  scrglobulins, 
fibrinogen,  and  fibrin  ferment.  The  two  last-mentioned  bodies  occur  only 
in  very  small  amounts ;  therefore  the  chyle  and  lymph  coagulate  slowly  (but 
spontaneously)  and  yield  but  little  fibrin.  Like  other  liquids  poor  in  fibrin 
ferment,  chyle  and  lymph  do  not  at  once  coagulate  completely,  but  repeated 
coagulations  take  place. 

The  extractive  bodies  seem  to  be  the  same  as  in  plasma.  Sugar  (or 
at  least  a  reducing  substance)  is  found  in  about  the  same  quantity  as  in  the 
blood-serum,  but  in  larger  quantities  than  in  the  blood;    this  depends  on 

1  Zeitschr.  f.  Biologie,  36. 

211 


212  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

the  fact  that  the  blood-corpuscles  contain  no  sugar.  The  glycogen  detected 
by  Dastre  *  in  the  lymph  occurs  only  in  the  leucocytes.  According  to 
Rohmann  and  Bial  lymph  contains  a  diastatic  enzyme  similar  to  that  in 
blood-plasma,  and  Lepine  2  has  found  that  the  chyle  of  a  dog  during 
digestion  has  great  glycolytic  activity.  The  amount  of  urea  has  been  deter- 
mined by  Wtjrtz3  as  0.12-0.28  p.  m.  The  mineral  bodies  appear  to  be  the 
same  as  in  plasma. 

As  form-elements  leucocytes  and  red  blood-corpuscles  are  common  to  both 
chyle  and  lymph.  Chyle  in  fasting  animals  has  the  appearance  of  lymph. 
After  fatty  food  it  is,  on  the  contrary,  milky,  due  partly  to  small  fat-glob- 
ules, as  in  milk,  and  partly,  to  the  greatest  extent,  to  finely  divided  fat. 
The  nature  of  the  fat  occurring  in  chyle  is  due  to  the  variety  existing  in 
the  food.  The  disproportionately  greater  part  consists  of  neutral  fat,  and 
even  after  feeding  with  large  quantities  of  free  fatty  acids  Mtjnk  4  found 
that  the  chyle  contained  chiefly  neutral  fat  with  only  small  amounts  of 
fatty  acids  or  soaps. 

The  gases  of  the  chyle  have  not  been  studied,  and  it  seems  that  the 
gases  of  an  entirely  normal  human  lymph  have  not  thus  far  been  investi- 
gated. The  gases  from  dog-lymph  contain  only  traces  of  oxygen  and. 
consist  of  37.4-53.1  per  cent  C02  and  1.6  per  cent  N,  calculated  at  0°  C,  and 
760  mm.  mercury.  The  chief  mass  of  the  carbon  dioxide  of  the  lymph 
seems  to  be  in  firm  chemical  combination.  Comparative  analyses  of  blood 
and  lymph  have  shown  that  the  lymph  contains  more  carbon  dioxide  than 
arterial,  but  less  than  venous  blood.  The  tension  of  the  carbon  dioxide 
of  lymph  is,  according  to  Pfluger  and  Strassburg,5  smaller  than  in  venous,, 
but  greater  than  in  arterial    blood. 

The  quantitative  composition  of  the  chyle  must  evidently  be  very  variable.9 

The  analyses  thus  far  made  refer  only  to  that  mixture  of  chyle  and  lymph 
which  is  obtained  from  the  thoracic  duct.  The  specific  gravity  varies 
between  1.007  and  1.043.  As  example  of  the  composition  of  human  chyle 
two  analyses  will  be  given.  The  first  is  by  Owen-Rees,  of  the  chyle 
of  an  executed  person,  and  the  second  by  Hoppe-Seyler,7  of  the  chyle  in 
a  case  of  rupture  of  the  thoracic  duct.  In  the  latter  case  the  fibrin  had 
previously  separated.     The  results  are  in  1000  parts. 

1  Compt.  rend,  de  soc.  biol.,  17,  and  Compt.  rend.,  120;  Arch,  de  Physiol.  (5),  7. 

2  Rohmann  and  Bial,  Pfliiger's  Arch.,  52,  53,  and  55;  L6pine,  Compt.  rend.,  110. 

3  Compt.  rend.,  49. 

*  Virchow's  Arch.,  80  and  123.  In  regard  to  the  analysis  of  the  fat  of  chyle,  see 
Erben.  Zeitschr.  f   physiol.  Chem.,  30. 

s  Ilammarsten,  "Die  Case  der  Hundelymphe,"  Arbeiten  aus  d.  physiol.  Anstalt  zu 
Leipzig,  1871;  Strassburg,  Pfliiger's  Arch.,  6. 

8  See  also  Panzer,  Zeitschr.  f.  physiol.  Chem.,  30. 

7  Owen-Rees,  cited  from  Hoppe-Seyler 's  Physiol.  Chem.,  595;  Hoppe-Seyler,  ibid.* 
597.     See  also  Carlier,  Brit.  Med.  Journ.,  1902,  175. 


THE  LYMPH.  213 

No.  1.  No.  2. 

Water 904 .8  940.72  water 

Solids 95.2  59.28  solids 

Fibrin Traces  

Albumin 70 . 8  38.67  albumin 

Fat 9.2  7.23  fat 

2 .  35  soaps 

( 0.83  lecithin 
■r,        .   .  .    ,     ,.  ir>  0  I  1.32  cholesterin 

Remaining  organic  bodies.  .     10.8  j  3. 03  alcohol  extractives 

[0.58  water  extractives 
_  ..  .    .  \  6.80  soluble  salts 

Salts 4-4  )  0.35  insoluble  salts 

The  quantity  of  fat  is  very  variable  and  may  be  considerably  increased 
by  partaking  food  rich  in  fats.  I.  Mtjnk  and  A.  Rosenstein  i  have  inves- 
tigated the  lymph  or  chyle  obtained  from  a  lymph  fistula  at  the  end  of  the 
upper  third  of  the  leg  of  a  girl  18  years  old  and  weighing  60  kg.,  and  the 
highest  quantity  of  fat  in  the  chylous  lymph  was  47  p.  m.  after  partaking 
of  fat.  In  the  starvation  lymph  from  the  same  patient  they  found  only 
0.6-2.6  p.  m.  fat.  The  quantity  of  soaps  was  always  small,  and  on  partaking 
of  41  grams  of  fat  the  quantity  of  soaps  was  only  about  g^  of  the  neutral 
fats. 

A  great  many  analyses  of  chyle  from  animals  have  been  made,  and 
they  chiefly  show  the  fact  that  the  chyle  is  a  liquid  with  a  very  changeable 
composition  which  stands  closely  related  to  blood-plasma,  but  with  the 
chief  difference  that  it  contains  more  fat  and  less  solids.  The  reader  is 
referred  to  special  works  for  these  analyses,  as,  for  example,  to  v.  Gorup- 
Besanez's  "Lehrbuch  der  physiologischen  Chemie,"  4th  edition. 

The  composition  of  the  lymph  is  also  very  changeable,  and  its  specific 
gravity  shows  about  the  same  variation  as  the  chyle.  In  the  following 
analyses,  1  and  2,  made  by  Gubler  and  Qtjevenne,  are  the  results  ob- 
tained from  lymph  from  the  upper  part  of  the  thigh  of  a  woman  aged  39; 
and  3,  made  by  v.  Scherer,  is  an  analysis  of  lymph  from  the  sac-like 
dilated  lymphatic  vessels  of  the  spermatic  cord.  Xo.  4  was  made  by 
C.  Schmidt,2  the  data  being  obtained  from  lymph  from  the  neck  of  a  colt. 
The  results  are  in  parts  per  1000. 

12  3            4 

Water 939.9  934. S  957.6  955.4 

Solids.... 60.1  65.2  42.4  44.6 

Fibrin 0.5  0.6  0.4           2.2 

Albumin 42.7  42.8  34.7/  

1  at,  cholesterin,  lecithin 3.8  9.2          [■  35.0 

Extractive  bodies 5.7  4.4  ....)  .... 

Salts 7.3  8.2  7.2           7.5 


1  Virchow's  Arch.,  123. 

2  Gubler  and  Quevenne,  cited  from  Hoppe-Seyler's  Physiol.  Chem.,  591;   Scherer, 
ibid.,  591;  C.  Schmidt,  ibid.,  592. 


214  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

The  salts  found  by  C.  Schmidt  in  the  lymph  of  the  horse  has  the  fol- 
lowing composition,  calculated  in  parts  per  1000  parts  of  the  lymph: 

Sodium  chloride 5 .  67 

Soda 1 .27 

Potash 0.16 

Sulphuric  acid 0 .  09 

Phosphoric  acid  united  with  alkalies 0 .  02 

Earthy  phosphates 0 .  26 

In  the  cases  investigated  by  Munk  and  Rosenstein  the  quantity  of 
solids  in  the  fasting  condition  varied  between  35.7  and  57.2  p.  m.  This 
variation  was  essentially  dependent  upon  the  extent  of  secretion,  so  that 
the  low  amount  coincides  with  a  more  active  secretion,  and  the  reverse  in 
the  other  case.  The  chief  portion  of  the  solids  consisted  of  proteids,  and 
the  relationship  between  globulin  and  albumin  was  as  1 : 2.4  to  4.  The 
mineral  bodies  in  1000  parts  lymph  (chylous)  was:  NaCl  5.83;  Na2C03  2.17; 
K2HPO40.28;  Ca3(P04)2  0.28;  Mg3(P04)2  0.09;  and  Fe(P04)  0.025. 

Under  special  conditions  the  lymph  may  be  so  rich  in  finely  divided  fat 
that  it  appears  like  chyle.  Such  lymph  has  been  investigated  by  Hensen 
in  a  case  of  lymph  fistula  in  a  ten-year-old  boy,  and  by  Lang  x  in  a  case  of 
lymph  fistula  in  the  left  upper  part  of  the  thigh  of  a  girl  of  seventeen. 
The  lymph  investigated  by  Hensen  varied  in  the  quantity  of  fat,  as  an 
average  of  nineteen  analyses,  between  2.8  and  36.9  p.  m.,  while  that  inves- 
tigated by  Lang  contained  24.85  p.  m.  of  fat. 

The  quantity  of  lymph  secreted  must  naturally  change  considerably 
under  various  conditions,  and  there  are  no  means  of  measuring  it.  The 
size  of  the  flow  of  lymph  is,  as  Heidenhain  suggests,  no  measure  of  the 
abundance  of  supply  of  nutritive  material  to  the  organs,  and  the  lymph- 
tubes  act  according  to  him  as  "drain- tubes,"  removing  the  excess  of  fluid 
from  the  lymph-fissures  as  soon  as  the  pressure  therein  rises  to  a  certain 
height.  Attempts  have  been  made  to  determine  the  quantity  of  lymph 
flowing  in  24  hours  in  the  thoracic  duct  of  animals.  According  to 
Heidenhain  the  quantity  averages  640  c.  c.  for  a  dog  weighing  10  kilos. 

Determinations  of  the  quantity  of  lymph  in  man  have  also  been 
attempted.  Noel-Paton  3  obtained  1  c.  c.  of  lymph  per  minute  from  the 
severed  thoracic  duct  of  a  patient  weighing  60  kilos.  The  quantity  in  the 
24  hours  cannot  be  calculated  from  this  amount.  In  the  case  of  Munk  and 
Rosenstein,  1134-1372  grams  chyle  was  collected  within  12-13  hours  after 
partaking  of  food.  In  the  fasting  condition  or  after  starving  for  18  hours 
they  found  50  to  70  grams  per  hour,  sometimes  120  grams  and  above,  espe- 
cially in  the  first  few  hours  after  powerful  muscular  exercise. 

Several  circumstances  have  a  marked  influence  on  the  extent  of  lymph 

1  Hensen,  Pfliiger's  Arch. ,  10;  Lang,  see  Maly 's  Jahresber.,  4. 

2  Journ.  of  Physiol.,  11. 


LYMPH  FORMATION.  215 

secretion.  During  starvation  less  lymph  is  secreted  than  after  partaking 
of  food.  Nasse  l  has  observed  in  dogs  that  the  formation  of  lymph  is 
increased  36  per  cent  more  after  feeding  with  meat  than  alter  feeding  with 
potatoes,  and  about  ">4  per  cent  more  than  after  24  hours'  deprivation  of 

food.  In  this  connection  mention  must  be  matfebf  the  important  observations 
of  Asher  and  Barbara  2  that  with  pure  protcid  diet  the  lymph  current 

is  increased  in  the  thoracic  cavity  and  also  that  the  increase  in  the  lymph 
secretion  runs  parallel  with  the  elimination  of  nitrogen  in  the  urine,  i.e., 
with  the  absorption  of  the  proteid  from  the  digestive  tract. 

An  increase  in  the  total  quantity  of  blood,  as  by  transfusion  of  blood,  also 
especially  on  preventing  the  flow  of  blood  by  means  of  ligatures,  cause-  an 
increase  in  the  quantity  of  lymph.  According  to  Heidenhain,  on  the 
contrary,  a  very  considerable  change  in  the  pressure  in  the  aorta  causes 
only  a  little  change  in  the  abundance  of  the  lymph-flow.  The  quantity  of 
lymph  may  be  raised  by  powerfully  active  and  passive  movements  of  the 
limbs  (Lesser).  Under  the  influence  of  curara  an  increase  of  the  lymph 
secretion  is  observed  (Paschutin,  Lesser3),  and  the  quantity  of  solids  in 
the  lymph  is  also  increased. 

The  bodies  inciting  lymph-flow,  the  so-called  hjmphagogues ,  are  of  espe- 
cially great  interest  and  they  may,  according  to  Heidenhain,4  be  divided 
into  two  different  chief  groups.  The  lymphagogues  of  the  first  series — 
extracts  of  crab-muscles,  blood-leech,  anodons,  liver  and  intestine  of  dogs, 
as  well  as  peptone  and  egg  albumin,  strawberry  extracts,  metabolic  products 
of  bacteria  and  others — cause  a  greatly  increased  secretion  of  lymph  without 
raising  the  blood  pressure,  and  in  this  way  the  blood-plasma  becomes 
poorer  in  proteids  and  the  lymph  richer  than  before.  For  the  formation 
of  this  lymph,  which  Heidenhain  designates  blood-lymph,  we  must  admit 
with  him  of  a  special  secretory  activity  of  the  capillary-wall  endothelium. 
The  lymphagogues  of  the  second  series,  such  as  sugar,  urea,  sodium  chloride, 
and  other  salts,  also  cause  an  abundant  lymph  formation.  The  blood, 
as  well  as  the  lymph,  thereby  becomes  richer  in  water.  This  increased 
amount  of  water  depends,  according  to  Heidenhaix,  upon  an  increased 
delivery  of  water  by  the  tissue-elements,  and  this  lymph  is  chiefly  tissue- 
lymph,  according  to  him.  Diffusion  is  no  doubt  of  great  importance  in 
the  formation  of  this  lymph,  but  the  secretory  activity  of  the  endothelium 
is  also  of  importance  at  least  for  certain  bodies,  such  as  sugar. 

'Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  593. 

2  The  works  of  Asher  and  collaborators,  Barbdra,  Gies  and  Busch,  upon  lymph 
formation  may  be  found  in  Zietschr.  f.  Biologic,  30,  37,  40. 

3  Lesser,  Arbeiten  aus  der  physiol.  Anstalt  zu  Leipzig,  Jahrgang  6;  Paschutin, 
ibv\,  7. 

4  Heidenhain,  Pfliiger's  Arch.,  49;  Hamburger,  Zeitschr.  f.  Biologie,  27  and  30. 
See  especially  Ziegler's  Beitr.  zur  path.  u.  zur  allg.  Pathol.,  14,  443;  also  Du  Bois- 
Reymond's  Arch.,  1S95  and  1890. 


21C  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

In  the  past  the  formation  of  lymph  was  explained  in  a  purely  physical 
way  by  the  united  action  of  filtration  from  the  blood  and  the  osmosis 
between  the  blood  and  tissue-fluid.  Later  Heidenhain  and  Hamburger 
ascribe  a  special  activity  to  the  capillary  endothelium  in  that  they  take 
part  in  the  formation  of  lymph  in  a  secretory  manner. 

Another  view  which  also  besides  the  physical  processes  is  of  especial 
physiological  moment  in  the  explanation  of  lymph  formation  was  sug- 
gested by  Asher  and  his  collaborators  (Barbera,  Gies  and  Busch). 
According  to  them  the  lymph  is  a  product  of  the  work  of  the  organs;  its 
amount  is  dependant  upon  an  increased  or  diminished  activity  of  the  organs 
and  the  lymph  is  therefore  a  measure  of  the  work  in  these.  The  close 
relation  between  lymph  formation  and  organ  work  has  also  been  shown 
for  several  organs,  especially  for  the  liver.  Starling  has  shown  that  after 
the  introduction  of  lymphagogues  of  the  first  series  chiefly  liver  lymph  is 
secreted,  which  he  claims  is  a  proof  against  Heidenhain 's  view,  and  he 
explains  the  increased  permeability  of  the  vessel  wall  by  the  fact  that  these 
bodies  have  a  poisonous  irritating  action.  On  the  contrary,  Asher  explains 
this  increased  lymph-flow  by  the  statement  that  the  substance  in  question — 
as  well  as  those  influences  which  incite  the  activity  of  the  liver — produces 
an  increased  formation  of  lymph  in  these  organs.  This  view  is.  supported 
by  the  experience  upon  the  action  of  lymphagogues  upon  blood  coagula- 
tion and  liver  activity  (Delezenne  and  others),  as  according  to  Gley  these 
bodies  have  at  the  same  time  a  lymphagogue  action  and  an  action  upon 
the  secretion  of  the  glands.  The  connection  between  organ  activity  and 
lymph  formation  has,  besides  the  above-mentioned  investigators,  also 
been  shown  by  others  upon  muscles  and  glands  (Hamburger,  Bainbridge1). 

The  extent  of  organ  work  certainly  essentially  influences  the  quantity  and 
properties  of  the  lymph.  Still  from  this  we  cannot  draw  any  positive  con- 
clusions as  to  whether  the  lymph  formation  is  brought  about  by  physico- 
chemical  processes  alone  or  if  in  this  process  a  specific,  not  closely  de- 
finable, secretory  force  is  at  work  at  the  same  time.  In  regard  to  this 
much-disputed  question  attention  must  be  called  in  the  first  place  to  the  fact 
that  the  important  works  of  Heidenhain,  Hamburger,  Lazarus-Barlow, 
and  others,  as  well  as  the  investigations  of  Asher  and  Gies  and  of  Mendel 
and  Hooker2  upon  the  lengthy  post-mortem  lymph-flow,  have  shown 
that  the  older  filtration  hypothesis  is  untenable.  That  the  part  played 
by  filtration  as  compared  to  that  of  the  osmotic  force  is  only  very  trivial 
has  been  conclusively  shown  by  the  adherents  of  the  physico-chemical 
theory  of  lymph  formation. 

Several  investigators  (Koranyi,  Starling,  Roth,  Asher,  and  others) 

1  In  regard  to  the  works  cited,  as  well  as  the  literature  upon  lymph  formation,  see 
Ellinger,  "Die  Bildung  der  Lymphe,"  Ergebnisse  des  Physiol.,  I,  Abt.  I,  1902. 

2  Amer.  Journ.  of  Physiol.,  7. 


TRANSUDATES  AND  EXUDATES.  217 

have  shown  clearly  that  the  work  in  the  glands  and  tissue-cells  must  cause  a 
difference  in  the  osmotic  pressure  upon  the  two  sides  of  the  capillary-walL 

That  this  is  so  follows  from  several  circumstances  and  also  from  the  fact 
that  iu  dissimilation  in  the  cells,  bodies  of  high  molecular  weight  are  split 
into  a  number  of  smaller  molecules,  which  latter,  either  directly,  if  they 
leave  the  cells  and  pass  into  the  tissue-fluid,  or  indirectly,  when  they  remain 
in  the  cells,  produce  an  increase  in  the  osmotic  tension  within  the  cells  and  in 
this  way  cause  a  taking  up  of  water  from  the  fluid  and  must  therefore 
increase  the  osmotic  pressure  of  the  tissue-fluids.  As  the  cells  can  by  syn- 
thesis build  up  highly  complex  constituents  from  simple  molecules,  and  as 
the  chief  products  of  catabolism  are  carbon  dioxide  and  water,  it  Is  diffi- 
cult to  explain  these  intricate  conditions.  Still,  irrespective  of  whatever 
view,  a  change  in  one  or  the  other  direction  in  the  osmotic  pressure  upon 
both  sides  of  the  capillary-wall  must  be  produced  hereby.  Whether  this 
and  other  physico-chemical  processes  are  alone  sufficient  to  explain  the 
lymph  formation  (Cohxsteix,  Ellixger)  remains  an  open  and  disputed 
question.1 

II.  Transudates  and  Exudates. 

The  serous  membranes  are  normally  kept  moistened  by  liquids  whose 
quantity  Is  only  sufficient  in  a  few  instances,  as  in  the  pericardial  cavity 
and  the  subarachnoidal  space,  for  a  complete  chemical  analyis  to  be  made 
of  them.  Under  diseased  conditions  an  abundant  transudation  may  take 
place  from  the  blood  into  the  serous  cavities,  into  the  subcutaneous  tissues, 
or  under  the  epidermis;  and  in  this  way  pathological  transudates  are 
formed.  Such  true  transudates,  which  are  similar  to  lymph,  are  gener- 
ally poor  in  form-elements  and  leucocytes,  and  yield  only  very  little  or 
almost  no  fibrin,  while  the  inflammatory  transudates,  the  so-called  exu- 
dates, are  generally  rich  in  leucocytes  and  yield  proportionally  more  fibrin. 
As  a  rule,  the  richer  a  transudate  is  in  leucocytes  the  closer  it  stands  to 
pus,  while  a  diminished  quantity  of  leucocytes  renders  it  more  nearly  like  a 
real  transudate  or  lymph. 

It  is  ordinarily  accepted  that  filtration  is  of  the  greatest  importance 
in  the  formation  of  transudates  and  exudates.  The  facts  coincide  with 
this  view  that  all  these  fluids  contain  the  salts  and  extractive  bodies 
occurring  in  the  blood-plasma  in  about  the  same  quantity  as  the  blood- 
plasma,  while  the  amount  of  proteids  is  habitually  smaller.  While  the 
different  fluids  belonging  to  this  group  have  about  the  same  quantities  of 
salts  and  extractive  bodies,  they  differ  from  one  another  chiefly  in  contain- 
ing differing  quantities  of  proteid  and  form-elements,  as  well  as  varying 


1  On  this  question  see  Ellinger,  "Die  Bildung  der  Lymphe,"  Ergebnisse  der  Thy- 
siologie,  I,  Abt.  1,  355. 


218  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

quantities  of  transformation  and  decomposition  products  of  these  latter — 
changed  blood-coloring  matters,  cholesterin,  etc.  The  correspondence  in 
the  amount  of  salts  and  extractive  bodies  present  in  the  blood  and  in 
transudates  supplies  just  as  little  proof  for  a  filtration  as  it  does  for  the  form- 
ation of  lymph;  but  still  it  cannot  be  doubted  for  other  reasons  that  fil- 
tration is  often  of  great  importance  in  the  formation  of  a  transudate.  To 
what  extent  filtration  is  active  in  the  perfectly  normal  vascular  wall  cannot 
be  answered. 

The  changed  permeability  of  the  capillary  walls  in  disease  is  a  second 
important  factor  in  the  formation  of  transudates.  The  circumstance  that 
the  greatest  quantity  of  proteid  occurs  in  transudates  in  inflammatory 
processes,  to  which  is  also  due  the  abundant  quantity  of  form-elements  in 
such  transudates,  has  been  explained  by  this  hypothesis.  The  greater 
quantity  of  proteid  in  the  transudates  in  formative  irritation  is  in  great 
part  explained  by  the  large  amount  of  destroyed  form-elements.  The 
interesting  observation  made  by  Paijkull,1  that  in  such  cases  in  which  an 
inflammatory  irritation  has  taken  place  the  fluid  contains  nucleoalbumin 
(or  nucleoproteids?),  while  these  substances '  do  not  occur  in  transudates 
in  the  absence  of  inflammatory  processes,  can  be  explained  by  the  pres- 
ence of  form-elements.  Still,  such  a  phosphorized  protein  substance  does 
not  occur  in  all  inflammatory  exudates. 

As  the  secretory  importance  of  the  capillary  endothelium  has  been  made 
probable  by  the  investigations  of  Heidenhain,  it  is  a  priori  to  be  expected 
that  an  abnormally  increased  secretory  activity  of  the  endothelium  is  a 
cause  of  transudates.  Those  observations  which  substantiate  such  an 
assumption  can  also  be  explained  just  as  well  by  assuming  a  changed 
permeability  of  the  capillary-walls. 

The  varying  quantities  of  proteid  observed  by  C.  Schmidt  2  in  the 
tissue-fluids  in  different  vascular  regions  can  perhaps  be  explained  by  the 
different  condition  of  the  capillary  endothelium.  For  example,  the  amount 
of  proteid  in  the  pericardial,  pleural,  and  peritoneal  fluids  is  con- 
siderably greater  than  in  those  fluids  which  are  found  in  the  subarach- 
noidal space,  in  the  subcutaneous  tissues,  or  in  the  aqueous  humor, 
which  are  poor  in  proteid.  The  condition  of  the  blood  also  greatly  affects 
the  transudates,  for  in  hydrsemia  the  amount  of  proteid  in  the  transudate 
is  very  small.  With  the  increase  in  the  age  of  a  transudate,  of  a  hydrocele 
fluid  for  instance,  the  quantity  of  proteid  is  increased,  probably  by  resorp- 
tion of  water,  and  indeed  exceptional  cases  may  occur  in  which  the  amount 
of  proteid,  without  any  previous  hemorrhage,  is  even  greater  than  in  the 
blood-serum. 

The  proteids  of  transudates  are  chiefly  seralbumin,  serglobulin,  and  a 

1  See  Maly's  Jahresber.,  22. 

2  Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  607. 


TRANSUDATES  AND  EXUDATES.  219 

little  fibrinogen.  Proteoses  and  peptones  do  not  occur,  with  perhaps  the 
cerebrospinal  fluid  as  an  exception,  and  in  those  cases  where  an  autolysis  has 
taken  place  in  the  liquid.1  The  non-inflammatory  transudates  do  not  as 
a  rule  coagulate  spontaneously,  or  very  slowly.  On  the  addition  of  blood 
or  blood-serum  they  coagulate.  Inflammatory  exudates  coagulate  spon- 
taneously, and  Paijkull  has  shown  that  these  often  contain  nucleoproteid 
(or  nucleoalbumin).  In  inflammatory  exudates  a  protein  substance  has 
been  habitually  observed  which  is  precipitated  by  acetic  acid,  but  which 
does  not  occur  in  transudates,  or  only  in  very  small  quantities.  This  sub- 
stance, which  was  observed  and  studied  by  Moritz,  Staeheun,  and 
Umber,  is  free  from  phosphorus  and  is  called  serosamucin  by  Umbeb, 
although  it  only  yields  very  little  reducing  carbohydrate.  According  to 
Joachim  2  it  is  only  a  part  of  the  globulin,  and  further  investigation  is 
very  desirable.  Mucoid  substances,  which  were  first  observed  by  Hammar- 
STEN  in  certain  cases  of  ascites  without  complication  with  ovarial  tumors, 
seem  according  to  Paijkull  3  to  be  a  regular  constituent  of  transudates 
as  a  cleavage  product  of  a  more  complicated  substance. 

There  are  numerous  investigations  on  the  relationship  between  globu- 
lin and  seralbumin,  and  Joachim  has  recently  determined  the  relationship 
between  euglobulin  and  the  total  globulin.  No  conclusive  results  can  be 
drawn  from  these  determinations.  The  relationship  between  globulin  and 
seralbumin  varies  very  much  in  different  cases,  but,  as  Hoffmann  and 
Pigeaud  4  have  shown,  the  variation  is  in  each  case  the  same  as  in  the  blood- 
serum  of  the  individual. 

The  specific  gravity  runs  rather  parallel  with  the  quantity  of  proteid. 
The  varying  specific  gravity  has  been  suggested  as  a  means  of  differentiation 
between  transudates  and  exudates  by  Reuss,5  as  the  first  often  show  a 
specific  gravity  below  1015-1010,  while  the  others  have  a  specific  gravity 
of  1018  or  above.     This  rule  holds  good  in  many  but  not  in  all  cases. 

The  gases  of  the  transudates  consist  of  carbon  dioxide  besides  small 
amounts  of  nitrogen  and  traces  of  oxygen.  The  tension  of  the  carbon 
dioxide  is  greater  in  the  transudates  than  in  the  blood.  When  mixed 
with  pus  the  amount  of  carbon  dioxide  is  decreased. 

The  extractives  are,  as  above  stated,  the  same  as  in  the  blood-plasma; 
but  sometimes  extractive  bodies  occur,  such  as  allantoin  in  dropsical  fluids 

1  Umber,  Munch,  med.  Wochenschr. ,  1902,  and  Berlin,  klin.  Wochenschr.,  1903. 
J  Paijkull,  1.   c. ;  Moritz,  Munch,  med.  Wochenschr.,  1903;   Staehelin,  ibid.,  1902; 
Umber,  Zeitschr.  f.  klin.  Med.,  48;  Joacbim,  Pfluger's  Arch.,  93. 

3  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  15;  Paijkull,  1.  c.  See  also  Young, 
Uber  das  Mucoid  der  Ascitesflussigkeit,  Inaug.  Diss.  Zurich,  1901. 

4  Joachim,  1.  c. ;  Hoffmann,  Arch.  f.  exp.  Path.  u.  Pharm.,  16;  Pigeaud,  see  Maly's 
Jahresber.,  16. 

6  Reuss,  Deutsch.  Arch.  f.  klin.  Med.,  28.    See  also  Otto,  Zeitschr.  f.  Heilkunde,  17. 


220  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

(Moscatelli  l),  which  have  not  been  detected  in  the  blood.  Urea  seems 
to  occur  in  ver}r  variable  amounts.  Sugar  also  occurs  in  transudates,  but 
it  is  not  known  to  what  extent  the  reducing  power  is  due,  as  in  blood- 
serum,  to  other  bodies.  A  reducing,  non-fermentable  substance  has  been 
found  by  Pickardt  in  transudates.  The  sugar  is  generally  dextrose,  but 
laevulose  seems  to  have  been  found 2  in  several  cases.  Sarcolactic  acid  has 
TDeen  found  by  C.  Kulz  3  in  the  pericardial  fluid  from  oxen.  Succinic  acid 
has  been  found  in  a  few  cases  in  hydrocele  fluids,  while  in  other  cases  it 
is  entirely  absent.  Leucin  and  tyrosin  have  been  found  in  transudates 
from  diseased  livers  and  in  pus-like  transudates  which  have  undergone 
decomposition,  and  after  autolysis.  Among  other  extractives  found  in 
transudates  must  be  mentioned  uric  acid,  xanthine,  creatine,  inosite,  and 
jiyrocatechin  (?). 

As  above  stated,  irrespective  of  the  varying  number  of  form-elements 
contained  in  the  different  transudates,  the  quantity  of  proteid  is  the  most 
characteristic  chemical  distinction  in  the  composition  of  the  various  trans- 
udates; therefore  a  quantitative  analysis  is  only  of  importance  in  so  far 
as  it  considers  the  quantity  of  proteid.  On  this  account,  in  the  following 
quantitative  composition,  chief  stress  will  be  put  on  the  quantity  of  proteid. 

Pericardial  Fluid.  The  quantity  of  this  fluid  is  also,  under  certain 
physiological  conditions,  so  large  that  a  sufficient  quantity  for  chemical 
investigation  was  obtained  from  a  person  who  had  been  executed.  This 
fluid  is  lemon-yellow  in  color,  somewhat  sticky,  and  yields  more  fibrin  than 
other  transudates.  The  amount  of  solids,  according  to  the  analyses  per- 
formed by  v.  Gorup-Besanez,  Wachsmuth,  and  Hoppe-Seyler,4  is 
37.5-44.9  p.  m.,  and  the  amount  of  proteid  is  22.8-24.7  p.  m.  The  analysis 
made  by  Hammarsten  of  a  fresh  pericardial  fluid  from  a  young  man  who 
had  been  executed  yielded  the  following  results,  calculated  in  1000  parts  by 
weight : 


Solids 

. ..     39.15 

.  Fibrin 

.  Globulin. .  .  . 

NaCl 

Soluble  salts 

28.60< 
8.60 

0.31 

5.95 

22.34 

. ..     7.28 

0.15 

2.00 

Friend  5  has  found  nearly  the  same  composition  for  a  pericardial  fluid 
from  a  horse,  with  the  exception  that  this  liquid  was  relatively  richer  in 

1  Zeitechr.  f.  physiol.  Chem.,  13. 

2  Pickardt,  Berl.  klin.  Wochenschr.,  1897.     See  also  Rotmann,  Munch,  med.  Woch- 
enschr.,  1898;   Neuberg  and  Strauss,  Zeitschr.  f.  physiol.  Chem.,  36. 

Zeitechr.  f.  Biologie,  :J2. 

4  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4,  Aufl.,  401;    Wachsmuth,  Vir- 
chow's  Arch.,  7;  Hoppe-Seyler,  Physiol.  Chem.,  605. 

5  Halliburton,  Text-book  of  Chem.  Physiol.,  etc.,  347.     London,  1891. 


PLEURAL  AND  PERITONEAL  FLUIDS.  221 

globulin.  The  ordinary  statement  that  pericardial  fluids  are  richer  in 
fibrinogen  than  other  transudates  is  hardly  based  on  sufficient  proof.  In 
a  case  of  chylopericardium,  which  was  probably  due  to  the  rupture  <>f  a 

chylous  vessel  or  caused  by  a  capillary  exudation  of  chyle  because  of  Stop- 
page, Hasebroek  '  found  in  1000  parts  of  the  fluid  103.61  parts  solids, 
7:i.7(.>  parts  proteids.  10.77  parts  fat.  3.34  parts  cholesterin,  1.77  parts 
Lecithin,  and  9.3  1  parts  salts. 

The  pleural  fluid  occurs  under  physiological  conditions  in  such  si  nail 
quantities  that  a  chemical  analysis  of  the  same  cannot  be  made.  Under 
pathological  conditions  this  fluid  may  show  very  variable  properties.  In 
certain  cases  it  is  nearly  serous,  in  others  again  sero-fibrinous,  and  in  others 
similar  to  pus.  There  is  a  corresponding  variation  in  the  specific  gravity 
and  the  properties  in  general.  If  a  pus-like  exudate  is  kept  closed  for  a 
long  time  in  the  pleural  cavity,  a  more  or  less  complete  maceration  and 
solution  of  the  pus-corpuscles  is  found  to  take  place.  The  ejected  yellowish- 
brown  or  greenish  fluid  may  then  be  as  rich  in  solids  as  the  blood-serum; 
and  an  abundant  flocculent  precipitate  of  a  nucleoalbumin  or  nucleopro- 
teid  (the  pyin  of  early  writers)  may  be  obtained  on  the  addition  of  acetic 
acid.     This  precipitate  is  soluble  with  difficulty  in  an  excess  of  acetic  acid. 

Numerous  analyses,  by  many  investigators,2  of  the  quantitative  composi- 
tion of  pleural  fluids  under  pathological  conditions  are  at  hand.  From 
these  analyses  we  learn  that  in  hydrothorax  the  specific  gravity  is  lower  and 
the  quantity  of  proteid  less. than  in  pleuritis.  In  the  first  case  the  specific 
gravity  is  generally  less  than  1015,  and  the  quantity  of  proteid  10-30 
p.  m.  In  acute  pleuritis  the  specific  gravity  is  generally  higher  than  1020, 
and  the  quantity  of  proteid  30-65  p.  m.  The  quantity  of  fibrinogen, 
which  in  hydrothorax  is  about  0.1  p.  m.,  may  amount  to  more  than  1  p.  m. 
in  pleuritis.  In  pleurisy,  with  an  abundant  gathering  of  pus,  the  specific 
gravity  may  rise  even  to  1030,  according  to  the  observations  of  Ham.mah- 
stf.n.  The  quantity  of  solids  is  often  60-70  p.  m.,  and  may  be  even  more 
than  90-100  p.  m.  (Hammarsten).  Mucoid  substances  have  also  been 
detected  in  pleural  fluids  by  Pai.ikull.  Cases  of  chylous  pleurisy  are  also 
known;  in  such  a  case  Mehu3  found  17.93  p.  m.  fat  and  cholesterin  in 
the  fluid. 

The  quantity  of  peritoneal  fluid  is  very  small  under  physiological  condi- 
tions. The  investigations  refer  only  to  the  fluid  under  diseased  conditions 
{dropsical  or  ascitic  fluid).  The  color,  transparency,  and  consistency  of 
these  may  vary  greatly. 

1  Zeitschr.  f.  physiol.  Chem.,  12. 

7  See  the  works  of  Mehu,  Runeberg,  F.  Hoffmann,  Reuss,  Xeuenkirchen,  all  of 
which  are  cited  in  Bernheim's  paper  in  Yirehow's  Arch.,  131,  274.  See  also  Paijkull, 
1.  c,  and  Halliburton's  Text-book,  346;  Joachim,  1.  c. 

3  Arch.  gen.  de  med.,  1886,  2,  cited  from  Maly's  Jahresber.,  16. 


222  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

In  cachectic  conditions  or  a  hydraemic  condition  of  the  blood  the  fluid 
has  little  color,  is  milky,  opalescent,  watery,  does  not  coagulate  spon- 
taneously, has  a  very  low  specific  gravity,  1005-1010-1015,  and  is  nearly 
free  from  form-elements.  The  ascitic  fluid  in  portal  stagnation,  or  gen- 
erally in  venous  congestion  has  a  low  specific  gravity  and  ordinarily  less 
than  20  p.  m.  proteid,  although  in  certain  cases  the  quantity  of  proteid 
may  rise  to  35  p.  m.  In  carcinomatous  peritonitis  it  may  have  a  cloudy, 
dirty-gray  appearance,  due  to  its  richness  in  form-elements  of  various  kinds. 
The  specific  gravity  is  then  higher,  the  quantity  of  solids  greater,  and  it 
often  coagulates  spontaneously.  In  inflammatory  processes  it  is  straw-  or 
lemon-yellow  in  color,  somewhat  cloudy  or  reddish,  due  to  leucocytes  and 
red  blood-corpuscles,  and  from  great  richness  in  leucocytes  it  may  appear 
more  like  pus.  It  coagulates  spontaneously  and  may  be  relatively  richer  in 
solids.  It  contains  regularly  30  p.  m.  or  more  proteid  (although  exceptions 
with  less  proteid  occur),  and  may  have  a  specific  gravity  of  1.030  or  above. 
By  rupture  of  a  chylous  vessel  the  dropsical  fluid  may  be  rich  in  very  finely 
emulsified  fat  (chylous  ascites).  In  such  cases  3,86-10.30  p.  m.  fat  has 
been  found  in  the  dropsical  fluid  (Guinochet,  Hay  x),  and  even  17-43  p.  m. 
has  been  found  by  Minkowski  . 

As  first  shown  by  Gross,  an  ascitic  fluid  may  have  a  chylous  appearance 
without  the  presence  of  fat,  i.e.,  pseudochylous.  The  reason  for  the  chylous 
properties  of  a  transudate  is  not  known,  although  numerous  investigators 
such  as  Gross,  Bernert,  Mosse,  Strauss  2  studied  the  subject;  several 
observations,  however,  seem  to  show  that  it  is  connected  with  the  amount  of 
lecithin  contained  therein. 

By  admixture  of  ascitic  fluid  with  the  fluid  from  an  ovarian  cyst  the 
former  may  sometimes  contain  pseudomucin  (see  Chapter  XIII).  There 
also  are  cases  in  which  the  ascitic  fluid  contains  mucoids  which  may  be  pre- 
cipitated by  alcohol  after  removal  of  the  proteids  by  coagulation  at  boiling 
temperature.  Such  mucoids,  which  yield  a  reducing  substance  on  boil- 
ing with  acids,  have  been  found  by  Hammarsten  in  tuberculous  peri- 
tonitis and  in  cirrhosis  hepatis  syphilitica  in  men.  According  to  the 
investigations  of  Paijkull  these  substances  seem  to  occur  often  and  perhaps 
habitually  in  the  ascitic  fluids. 

As  the  quantity  of  proteid  in  ascitic  fluids  is  dependent  upon  the  same 
circumstances  as  in  other  transudates  and  exudates,  it  is  sufficient  to  give 
the  following  example  of  the  composition,  taken  from  Bernheim's3 
treatise.    The  results  are  expressed  in  1000  parts  of  the  fluid : 


1  Guinochet,  see  Strauss,  Arch,  de  Physiol.,  18.     See  Maly's  Jahresber.,  16,  475. 

2  Gross,  Arch.  f.  exp.  Path.  u.  Pharm.,  44;    Bernert,  ibid.,  49;    Mosse,  Leyden'a 
Festschrift,  1901;  Strauss,  cited  in  Biochem.  Centralbl.,  1,  437. 

3  L.  c.     As  it  was  impossible  to  derive  mean  figures  from  those  given  by  Bernheim„ 
the  author  has  given  the  maximum  and  minimum  of  the  averages  given  by  him. 


HYDROCELE,  SPERMATOCELE,  CEREBROSPINAL  FLUID.        223 

Max.  Min.  Mean. 

Cirrhosis  of  the  liver 34 . 5  5.6  9 .  69—21 .  06 

Bright'fl  disease 16.11  10.10  5.6  —10.36 

Tuberculous  and  idiopathic  peritonitis.  .  .   55.8  18.72  30.7  — 37.95 

Carcinomatous  peritonitis 54.20  27.00  35. 1  —58.96 

Joachim  found  the  highest  relative  globulin  amounts  and  lowest  albumin 
results  in  cirrhosis;  in  carcinoma  on  the  contrary  the  lowest  globulin  and  the 
highest  albumin.  The  values  in  cardiac  stagnation  stand  between  the  cirrhosis 
and  carcinoma. 

Urea  has  also  been  found  in  ascitic  fluids,  sometimes  only  as  traces,  some- 
times in  larger  quantities  (4  p.  m.  in  albuminuria),  also  uric  acid,  allantoin  in 
cirrhosis  of  the  liver  (Moscatelli),  xanthine,  creatine,  cholesterin,  sugar,  diaatalic 
and  proteolytic  enzymes,  and  according  to  Hamburger  '  also  a  lipase. 

Hydrocele  and  Spermatocele  Fluids.  These  fluids  essentially  differ 
from  each  other  in  various  ways.  The  hydrocele  fluids  are  generally  colored 
light  or  dark  yellow,  sometimes  brownish  with  a  shade  of  green.  They 
have  a  relatively  higher  specific  gravity,  1.016-1.026,  with  a  variable  but 
generally  higher  amount  of  solids,  an  average  of  60  p.  m.  They  sometimes 
coagulate  spontaneously,  sometimes  only  after  the  addition  of  fibrin  ferment 
or  blood.  They  contain  leucocytes  as  chief  form-elements.  Sometimes 
they  contain  smaller  or  larger  amounts  of  cholesterin  crystals . 

The  spermatocele  fluids,  on  the  contrary,  are  as  a  rule  colorless,  thin, 
cloudy  like  water  mixed  with  milk.  They  sometimes  have  an  acid  reaction. 
They  have  a  lower  specific  gravity,  1.006-1.010,  a  lower  amount  of  solids — 
an  average  of  about  13  p.  m. — and  do  not  coagulate  either  spontaneously 
or  after  the  addition  of  blood.  They  are,  as  a  rule,  poor  in  proteid  and 
contain  spermatozoa,  cell-detritus,  and  fat-globules  as  form-constituents.  To 
show  the  unequal  composition  of  these  two  kinds  of  fluids  we  will  give  the 
average  results  (calculated  in  parts  per  1000  parts  of  the  fluid)  of  17  anal- 
yses of  hydrocele  fluids  and  4  of  spermatocele  fluids  made  by  Hammarsten.2 

Hydrocele.  Spermatocele. 

Water 938.85  986.83 

Solids 61.15  13.17 

Fibrin 0 .  59            

Globulin 13.25  0.59 

Seralbumin 35.94  1.82 

Ether  extractive  bodies 4 .02  ) 

Soluble  salts 8.60>  10.76 

Insoluble  salts 0 .  66 ) 

In  the  hydrocele  fluid  traces  of  urea  and  a  reducing  substance  have  been 
found,  and  in  a  few  cases  also  succinic  acid  and  inosite.  A  hydrocele  fluid  may, 
according  to  Devillaro,3  sometimes  contain  paralbumin  or  metalbumin  (?). 
Cases  of  chylous  hydrocele  are  also  known. 

Cerebrospinal  Fluid.  The  cerebrospinal  fluid  is  thin,  water-clear,  of 
low  specific  gravity,  1.007-1.008.     The  spina  bifida  fluid  is  very  poor  in 

1  Arch.  f.  (Anat.  u.)  Physiol.,  1900,  433. 

2  Upsala  Lakaref.  Forh.,  14,  and  Maly's  Jahresber.,  8,  347. 
8  Bull.  soc.  chixn.,  49,  617. 


224  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

solids,  8-10  p.  m.,  with  only  0.19-1.6  p.  m.  proteid.  The  fluid  of  chronic 
hydrocephahis  is  somewhat  richer  in  solids  (13-19  p.  m.)  and  proteids. 
According  to  Halliburton  the  proteid  of  the  cerebrospinal  fluid  is  a 
mixture  of  globulin  and  proteose;  occasionally  some  peptone  occurs,  and 
more  rarely,  in  special  cases,  seralbumin  appears.  The  statements  of 
Halliburton  on  the  occurrence  of  proteose  do  not  coincide  with  the 
observations  of  other  investigators  (Panzer,  Salkowski  *).  In  general 
paralysis  where,  according  to  Halliburton  and  Mott,2  choline  and  poison- 
ous products  from  the  brain  pass  into  the  fluid,  it  also  contains  nucleo- 
proteid.  Dextrose,  or  at  least  a  fermentable  sugar,  occurs  habitually  in  the 
cerebrospinal  fluid,  while  the  statements  of  Halliburton  as  to  the  occur- 
rence of  a  substance  similar  to  pyrocatechin  could  not  be  substantiated  by 
Nawratzki  3  and  hence  this  substance  does  not  exist  in  all  cerebrospinal 
fluids.  Urea  occurs  in  cerebrospinal  fluids  but  not  always.  The  variable 
relationship  between  potassium  and  sodium  4  is  probably  due,  according 
to  Salkowski,  to  the  absence  or  presence  of  fever  during  the  formation 
of  the  exudate;  the  amount  of  potassium  is  high  in  the  acute  cases  and  low 
in  the  chronic  ones.  According  to  Cavazzani,5  who  has  especially  studied 
the  cerebrospinal  fluids,  the  alkalinity  of  these  fluids  is  considerably  less 
than  the  blood  and  independent  of  this  last  fluid.  For  this  and  several 
other  reasons  Cavazzani  draws  the  conclusion  that  the  cerebrospinal 
fluid  is  formed  by  a  true  secretory  process. 

Aqueous  Humor.  This  fluid  is  clear,  alkaline  towards  litmus,  and  has 
a  specific  gravity  of  1.003-1.009.  The  amount  of  solids  is  on  an  average 
13  p.  m.  and  the  amount  of  proteids  only  0.8-1.2  p.  m.  The  proteid  con- 
sists of  seralbumin  and  globulin  and  very  little  fibrinogen.  According  to 
Gruenhagen  it  contains  paralactic  acid,  another  dextrogyrate  substance, 
and  a  reducing  body  which  is  not  similar  to  dextrose  or  dextrin.  Pautz  6 
found  urea  and  sugar  in  the  aqueous  humor  of  oxen. 

Blister-fluid.  The  content  of  blisters  caused  by  burns,  and  of  vesicator 
blisters  and  the  blisters  of  the  pemphigus  chronicus,  is  generally  a  fluid 
rich  in  solids  and  proteids  (40-65  p.m.).  This  is  especially  true  of  the 
contents  of  vesicatory  blisters.  In  a  burn-blister  K.  Morner7  found  50.31 
]>.  in.  proteids,  among  which  were  13.59  p.  m.  globulin  and  0.11  p.  m.  fibrin. 
The  fluid  contains  a  substance  which  reduces  copper  oxide  but  no  pyro- 

1  Halliburton's  Text-book,  355-361;  Panzer,  Wien.  klin.  Wochenschr.,  1899;  Sal- 
kowski, Jaffe'  Festschrift,  265. 

2  Phil.  Transact.  Roy.  Soc.  London,  Series  B,  191. 

3  Zeitschr.  f.  physiol.  chem.,  23.     See  also  Rossi,  ibid.,  39  (literature). 

■  Salkowski,   1.    c.     New   quantitative  analyses  of  cerebrospinal  and  hydro- 
cephalus fluids  may  be  found  in  the  cited  works  of  Nawratzki,  Panzer  and  Salkowski. 
5  See  Maly's  Jahresber.,  22,  346,  and  Centralbl.  f.  Physiol,  15,  216. 
'Gruenhagen,  Pfliiger's  Arch.,  43;   Pautz,  Zeitschr.  f.  Biologie,  31. 
7  Skand.  Arch.  f.  Physiol.,  5. 


SYNOVIAL  FLUID.  225 

catechin.  The  fluid  of  the  pemphigus  is  alkaline  in  reaction.  A  wound 
secretion  collected  by  Libblein1  under  aseptic  conditions  was  alkaline  in 
reaction  and  contained  less  proteid  than  the  blood-serum.  It  formed 
a  slight  fibrin  clol  and  only  contained  proteoses  at  first  or  at  the  beginning 
of  the  abscess  formation.  As  the  wound  healed  the  relationship  between 
the  globulin  and  albumin  changed,  and  on  the  third  day  of  the  healing 
the  quantity  of  albumin  was  at  least  nine  tenths  of  the  total  proteid. 

The  fluid  of  subcutaneous  oedema.  This  is,  as  a  rule,  very  poor  in 
solids,  purely  serous,  does  not  contain  fibrinogen,  and  has  a  specific  gravity 
of  1.005-1.013.  The  quantity  of  proteids  is  in  most  cases  lower  than  10 
p.  m., — according  to  Hoffmann  1-8  p.  m., — and  in  serious  affections  of 
the  kidneys,  generally  with  amyloid  degeneration,  less  than  1  p.  m.  has  been 
shown  (Hoffman x  2).  The  cedematous  fluid  also  habitually  contains  urea, 
1-2  p.  m.,  and  also  sugar. 

The  fu'id  of  the  tapeworm  cyst  is  related  to  the  transudates  and  poor  in 
proteids.  It  is  thin  and  colorless,  and  has  a  specific  gravity  of  1.005-1.015.  The 
quantity  of  solids  is  14-20  p.  m.  The  chemical  constituents  are  sugar  (2.5  p.  m.), 
inosite,  traces  of  urea,  creatine,  succinic  acid,  and  salts  (8.3-9.7  p.  m.).  Proteids 
are  only  found  in  traces,  and  then  only  after  an  inflammatory  irritation.  In  the 
last-mentioned  case  7  p.  m.  proteids  have  been  found  in  the  fluid. 

The  Synovial  Fluid  and  Fluid  in  Synovial  Cavities  around  Joints,  etc. 
The  synovia  is  hardly  a  transudate,  but  it  is  often  treated  as  an  appendix 
to  the  transudates. 

The  synovia  is  an  alkaline,  sticky,  fibrous,  yellowish  fluid  which  is 
cloudy,  from  the  presence  of  cell-nuclei  and  remains  of  destroyed  cells,  but 
is  also  sometimes  clear.  It  contains  also,  besides  proteids  and  salts,  a  sub- 
stance similar  to  mucin  in  physical  properties.  The  nature  of  this  mucin- 
like  constituent  of  physiological  synovial  fluids  has  not  been  determined. 
Hammarsten  has  found  a  mucin-like  substance  in  pathological  synovial 
fluid,  but  it  was  not  true  mucin.  It  acts  like  a  nucleoalbumin  or  a  nucleo- 
proteid,  and  yielded  no  reducing  substance  when  boiled  with  acid.  Sal- 
kowski  3  also  found  a  mucin-like  substance  in  a  pathological  synovial 
fluid,  which  was  neither  mucin  nor  nucleoalbumin.  He  called  the  sub- 
stance ' '  synovin. ' ' 

The  composition  of  synovia  is  not  constant,  but  varies  in  rest  and  in 
motion.  In  the  last-mentioned  case  the  quantity  of  fluid  is  less,  but  the 
amount  of  the  mucin-like  body,  proteids,  and  of  the  extractive  bodies  is 
greater,  while  the  quantity  of  salts  is  diminished.  This  may  be  seen  from 
the  following  analyses  by  Frerichs.4    The  figures  represent  parts  per  1000. 

1  Habilitationsschrift  Prag.,  1902.     Printed  by  H.  Laupp,  Tubingen. 

2  Deutsch.  Arch.  f.  klin.  Med.,  44. 

3  Hammarsten,  Maly's  Jahresber.,  12;  Salkowski,  Virchow's  Arch.,  131. 
*  Wagner's  Handwdrterbuch,  3,  Abth.  I,  463. 


226  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

I.  Synovia  from  II.  Synovia  from 

a  Stall-fed  Ox.  a  Field-fed  Ox. 

Water 969.9  948.5 

Solids 30.1  51.5 

Mucin-like  body 2.4  5.6 

Albumin  and  extractives 15.7  35 . 1 

Fat 0.6  0.7 

Salts 11.3  9.9 

The  synovia  of  new-born  babes  corresponds  to  that  of  resting  animals. 

The  fluid  of  the  bursse  mucosa?,  as  also  the  fluid  in  the  synovial  cavities 

around  joints,  etc.,  is  similar  to  synovia  from  a  qualitative  standpoint. 

III.    Pus. 

Pus  is  a  yellowish-gray  or  yellowish-green,  creamy  mass  of  a  faint  odor 
and  an  unsavory,  sweetish  taste.  It  consists  of  a  fluid,  the  pus-serum,  in 
which  solid  particles,  the  pus-cells,  swim.  The  number  of  these  cells  varies 
so  considerably  that  the  pus  may  at  one  time  be  thin  and  at  another  time 
so  thick  that  it  scarcely  contains  a  drop  of  serum.  The  specific  gravity, 
therefore,  may  also  greatly  vary,  namely,  between  1.020  and  1.040,  but 
ordinarily  it  is  1.031-1.033.  The  reaction  of  fresh  pus  is  generally  alkaline, 
but  it  may  become  neutral  or  acid  from  a  decomposition  in  which  fatty 
acids,  glycerophosphoric  acid,  and  also  lactic  acid  are  formed.  It  may 
become  strongly  alkaline  when  putrefaction  occurs  with  the  formation  of 
ammonia. 

In  the  chemical  investigation  of  pus,  the  pus-serum  and  the  pus-corpus- 
cles must  be  studied  separately. 

Pus-serum.  Pus  does  not  coagulate  spontaneously  nor  after  the  addi- 
tion of  defibrinated  blood.  The  fluid  in  which  the  pus-corpuscles  are  sus- 
pended is  not  to  be  compared  with  the  blood-plasma,  but  rather  with  the 
serum.  The  pus-serum  is  pale  yellow,  yellowish-green,  or  brownish-yellow, 
and  has  an  alkaline  reaction  towards  litmus.  It  contains,  for  the  most  part, 
the  same  constituents  as  the  blood-serum;  but  sometimes  besides  these — 
when,  for  instance,  the  pus  has  remained  in  the  body  for  a  long  time — it 
contains  a  nucleoalbumin  or  a  nucleoproteid  which  is  precipitated  by  acetic 
acid  and  soluble  with  great  difficulty  in  an  excess  of  the  acid  {pyin  of  the 
older  authors).  This  nucleoalbumin  seems  to  be  formed  from  the  hyaline 
substance  of  the  pus-cells  by  maceration.  The  pus-serum  contains,  more- 
over, at  least  in  many  cases,  no  fibrin  ferment.  According  to  the  analyses 
of  Hoppe-Seyler  !  the  pus-serum  contains  in  1000  parts: 

I.  II. 

Water 913.7  905.65 

Solids 86.3  94.35 

Proteids 63.23  77.21 

Lecithin 1.50  0.56 

Fat 0.26  0.29 

Cholesterin 0 .  53  0 .  87 

Alcohol  extractives 1 .  52  0 .  73 

Water  extractives 11 .  53  6 .  92 

Inorganic  salts 7 .  73  7 .  77 

1  Med.-chem.  Untersuch.,  490. 


PUS.  227 

The  ash  of  pus-serum  has  the  following  composition,  calculated  to  1000  parts 
of  the  serum: 

I.  II. 

NaCl 5.22  5  39 

N:.  .si  », 0.40  0.31 

NiiJII'd, 0.98  0.  Hi 

Na,(H.)3 0.49  1.13 

c.i    I't'.u 0.49  0.31 

Mg,(PO«), 0.19  0.12 

1  'i  )4  (in  excess) 0.05 

The  pus-corpuscles  are  generally  thought  to  consist  in  great  part  of 
emigrated  white  blood-corpuscles,  and  their  chemical  properties  have 
therefore  been  given  in  discussing  these.  We  consider  the  molecular  gran- 
ules, fat-globules,  and  red  blood-corpuscles  rather  as  casual  form-elements. 

The  pus-cells  may  be  separated  from  the  serum  by  centrifugal  force,  or 
by  decantation  directly  or  after  dilution  with  a  solution  of  sodium  sulphate 
in  water  (1  vol.  saturated  sodium-sulphate  solution  and  9  vols,  water),  and 
then  washed  by  this  same  solution  in  the  same  manner  as  the  blood-cor- 
puscles. 

The  chief  constituents  of  the  pus-corpuscles  are  proteids  of  which 
the  largest  proportion  seems  to  be  a  nucleoproteid  which  is  insoluble  in 
water  and  which  expands  into  a  tough,  slimy  mass  when  treated  with  a  10 
per  cent  common  salt  solution.  This  proteid  substance,  which  is  soluble  in 
alkali  but  is  quickly  changed  thereby,  is  called  Rovida's  hyaline  substance, 
and  the  property  of  the  pus  of  being  converted  into  a  slime-like  mass  by  a 
solution  of  common  salt  depends  on  this  substance.  Besides  this  substance 
the  pus-cells  contain  also  a  globulin  which  coagulates  at  48-49°  C,  as  well 
as  sirglobulin  (?),  seralbumin,  a  substance  similar  to  coagulated  proteid 
(Miescher),  and  lastly  peptone  or  proteose  (Hofmeister  *).  It  is  very 
remarkable  that  no  nucleohiston  or  histon  has  been  detected  in  the  pus- 
cells. 

There  is  also  found  in  the  protoplasm  of  the  pus-cells,  besides  the  pro- 
teids, lecithin,  cholesterin,  xanthine  bodies,  fat,  and  soaps.  Hoppe-Seyler  has 
found  cerebrin,  a  decomposition  product  of  a  protagon-like  substance,  in 
pus  (see  Chapter  XII).  Kossel  and  Freytag  2  have  isolated  from  pus  two 
substances,  pyosin  and  pyogenin,  which  belong  to  the  cerebrin  group  (see 
Chapter  XII).  Hoppe-Seyler3  claims  that  glycogen  appears  only  in  the 
living,  contractile  white  blood-cells  and  not  in  the  dead  pus-corpuscles. 
Several  other  investigators  have  nevertheless  found  glycogen  in  pus.  The 
cell-nucleus  contains  nuclcin  and  nucleoproteids. 

In  regard  to  the    occurrence  of  enzymes  in  the  pus-cells    it   must  be 

1  Miescher  in  Hoppe-Seyler 's  Med.-chem.  Untersuch.,  441;  Hofmeister,  Zeitschr.  f. 
physiol.  Chem.,  4. 

2  Ibid.,  17,  452. 

3  Hoppe-Seyler,  Physiol.  Chem.,  790. 


228  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

remarked  that  neither  thrombin  nor  prothrombin  are  found  therein,  although 
these  bodies  are  generally  considered  as  being  derived  from  the  leucocytes 
and  can  also  be  obtained  from  the  thymus  leucocytes.  The  occurrence 
in  the  pus-cells  also  of  a  proteolytic  enzyme  is  of  great  interest.  It  is 
not  only  important  for  the  intracellular  digestion  and  for  the  amount  of 
proteoses  in  the  pus-cell,  but  also  for  the  solution  of  the  fibrin  clot  and 
pneumonic  infiltrations  (F.  Muller,  0.  Simon  x). 

The  mineral  constituents  of  the  pus-corpuscles  are  potassium,  sodium, 
calcium,  magnesium,  and  iron.  A  part  of  the  alkalies  exists  as  chlorides,, 
and  the  remainder,  as  well  as  the  chief  part  of  the  other  bases,  exists  as 
phosphates. 

The  quantitative  composition  of  the  pus-cells  from  the  analyses  of 
Hoppe-Seyler  is  as  follows,  in  parts  per  1000  of  the  dried  substance: 

I.  II. 

Proteids 137.62) 

Nuclein 342 . 57 [■  685 . 85         673.69 

Insoluble  bodies 205 .  66 ) 

Lecithin [  i  aq  «q  75 .  64 

Fat )  14d-8d  75.00 

Cholesterin 74.00  72.83 

Cerebrin 51 .  99  )  jq2  04 


I 


Extractive  bodies 44 .  33 

MINERAL  SUBSTANCES  IN  1000  PARTS  OF  THE  DRIED  SUBSTANCE. 

NaCl 4.35 

Ca3(P04)2 2.05 

Mg3(P04)2 1.13 

FeP04 1 .  06 

P04 9.16 

Na 0.68 

K Traces  (?) 

Miescher  has  obtained  other  results  for  the  alkali  combinations,  namely,, 
potassium  phosphate  12,  sodium  phosphate  6.1,  earthy  phosphate  and  iron  phos- 
phate 4.2,  sodium  chloride  1.4,  and  phosphoric  acid  combined  with  organic  sub- 
stances 3.14-2.03  p.  m. 

In  pus  from  congested  abscesses  which  have  stagnated  for  some  time 
occurs  peptone  (proteose),  leucin,  and  tyrosin,  free  fatty  acids,  and  volatile 
fatty  acids,  such  as  formic  acid,  butyric  acid,  valerianic  acid.  There  are 
also  found  chondrin  (?)  and  glutin  (?),  urea,  dextrose  (in  diabetes),  bile- 
pigments  and  bile-acids  (in  catarrhal  icterus). 

As  more  specific  but  not  constant  constituents  of  the  pus  must  be  men- 
tioned the  following:  pyin,  which  seems  to  be  a  nucleoproteid  precipitable 
by  acetic  acid,  and  also  pyinic  acid  and  chlorrhodinic  acid,  which  have  been 
so  little  studied  that  they  cannot  be  more  fully  treated  here. 

In  many  cases  a  blue,  more  rarely  a  green,  color  has  been  observed  in 
the  pus.     This  depends  on  the  presence  of  micro-organisms  (Bacillus  pyo- 

1  Fr.  Muller,  Verhandl.  Nat.  Gesellsch.  zu  Basel,  1901 ;  O.  Simon,  Deutsch.  Arch. 
f.  klin.  Med.,  70. 


LYMPHATIC  GLANDS,  SI'LLLX,  ETC.  229 

cyaneus).  From  such  pus  Fordos  and  Lucke  *  have  isolated  a  crystalline 
blue  pigment,  pyoq/anin,  and  a  yellow  pigment,  pyoxanthose,  which  is  pro- 
duced from  the  first  by  oxidation. 

Appendix. 

LYMPHATIC   GLANDS,   SPLEEN,   ETC. 

The  Lymphatic  Glands.  The  cells  of  the  lymphatic  glands  are  found  to 
contain  the  protein  substances  occurring  generally  in  cells  (Chapter  V, 
pages  118  and  119).  According  to  Bang  2  they  also  contain  histon  nucleates 
(nuclcohiston),  but  in  smaller  amounts  and  of  a  different  variety  from  the 
better-studied  nuclcohiston  from  the  thymus  gland.  Proteoses  occur  as 
products  of  an  autolysis.  By  a  lengthy  autolysis  of  lymph-glands  Reh  3 
found  ammonia,  tyrosin,  leucin  (somewhat  less),  thymin,  and  uracil  among 
the  cleavage  products.  Besides  the  other  ordinary  tissue  constituents,  such 
as  collagen,  reticulin,  elastin,  and  nuclein,  there  occurs  in  the  lymphatic  glands 
also  cholcstcrin,  fat,  glycogen,  sarcolactic  acid,  xanthine  bodies,  and  leucin.  In 
the  inguinal  glands  of  an  old  woman  Oidtmann  found  714.32  p.  m.  water, 
284.5  p.  m.  organic  and  1.16  p.  m.  inorganic  substances.  In  the  cells  of 
the  mesenteral  lymphatic  glands  of  oxen  Bang  *  found  804.1  p.m.  water, 
195.9  p.  m.  solids,  137.8  total  proteins,  6.7  p.  m.  histon  nucleate,  10.6 
p.  m.  nucleoproteid,  47.6  p.  m.  bodies  soluble  in  alcohol,  and  10.5  p.  m. 
mineral  constituents. 

The  Thymus.  The  cells  of  this  gland  are  very  rich  in  nuclein  bodies 
and  relatively  poor  in  the  ordinary  proteids,  but  their  nature  has  not  been 
closely  studied.  The  chief  interest  is  attached  to  the  nuclein  substances. 
Kossel  and  Liliexfeld  first  prepared  from  the  water}'  extract  of  the 
gland,  by  precipitating  with  acetic  acid  and  then  further  purifying,  a  protein 
substance  which  has  been  generally  called  nuclcohiston.  By  the  action 
of  dilute  hydrochloric  acid  upon  nucleohiston  it  splits  according  to  these 
investigators  into  histon  and  leuconuclein.  The  leuconuclein  is  a  true 
nuclein;  hence  it  is  a  nucleic  acid  compound  with  proteid  which  is  relatively 
poor  in  proteid  and  rich  in  phosphorus.  The  more  recent  investigations 
of  Bang,  Malexgreau  and  Huiskamp  5  upon  nucleohiston  are  united 
that  this  nucleoproteid  is  not  a  unit  substance  but  a  mixture  of  at  least 

1  Fordos,  Compt.  rend.,  51  and  50;  Lucke,  Arch.  f.  klin.  Chirurg.,  3;  Boland,  Cen- 
tralbl.  f.  Bakt.  u.  Parasit.,  I,  25. 

2  Studier  over  Xucleoproteider.     Kristiania,  1902. 

3  Hofmeister's  Beitriige,  3. 
*L.  c. 

s  Lilienfeld,  Zeitschr.  f.  physiol.  Chem.,  IS;  Kossel,  ibid.,  30  and  31;  Bang,  ibid., 
30  and  31.  See  also  Arch.  f.  Math,  og  Xaturvidenskab.,  25,  Kristiania,  1902,  and 
Hofmeister's  Beitriige,  1  and  4;  Malengreau,  La  Cellule,  17  and  19;  Huiskamp,  Zeit- 
schr. f.  physiol  Chem.,  32,  34,  and  39. 


230  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

two  bodies.  The  views  of  the  mentioned  investigators  differ  quite  essen- 
tially from  one  another  as  to  the  nature  of  these  bodies,  but  this  is  partly 
due  to  the  different  methods  used  by  them  and  partly  upon  the  ready 
changeability  of  the  substances  in  question. 

Besides  the  real  nucleohiston,  B-nucleoalbumin  of  Malengreau,  Lilien- 
feld's  histon  contains  a  second  nucleoproteid  which  Bang  and  Huiskamp 
call  simple  nucleoproteid,  while  Malengreau  designates  it  A-nucleoalbumin. 
This  proteid,  which  only  contains  about  1  per  cent  phosphorus  and  which, 
is  possibly  identical  with  the  nucleoproteid  found  by  Lilienfeld  in  the 
thymus  yields  a  nuclein,  but  no  nucleic  acid  on  cleavage.  As  second  cleavage 
product  it  yields,  according  to  Malengreau,  the  A-histon,  which  can  be- 
readily  precipitated  by  magnesium  and  ammonium  sulphate  from  the 
ordinary  B-histon  of  the  thymus  gland.  The  occurrence  of  A-histon  in 
the  gland  has  been  verified'  by  Bang.  According  to  Bang  and  Huiskamp 
the  nucleoproteid  does  not  yield  any  histon;  it  only  yields  an  albuminate 
(Bang). 

The  true  nucleohiston,  which  is  much  richer  in  phosphorus  (the  calcium 
salt  containing,  according  to  Bang,  on  an  average  5.23  per  cent  P),  yields 
ordinary  histon  as  a  cleavage  product,  according  to  the  unanimous  opinion 
of  the  above-mentioned  investigators.  According  to  Bang,  whose  state- 
ments on  this  point  have  been  substantiated  by  Malengreau,  it  splits 
on  saturating  with  NaCl  into  nucleic  acid  and  histon  without  yielding  any 
other  proteid.  On  this  account  Bang  does  not  consider  this  body  as 
nucleohiston  in  the  ordinary  sense,  i.e.,  not  as  a  nucleoproteid,  but  as  a. 
histon  nucleate.  The  nucleohiston  behaves  like  an  acid,  whose  salts,, 
especially  the  calcium  salt,  has  been  closely  studied  by  Huiskamp.  On 
the  electrolysis  of  a  solution  of  nucleohiston  alkali  in  water  Huiskamp 
found  also  that  the  nucleohiston  collected  as  traces  at  the  anode,  and 
that  the  sodium  compound  is  therefore  ionized  in  the  solution.  The 
nucleic  acid-histon  calcium  combination  has  been  prepared,  it  seems,  in  a 
pure  state  by  Bang,  and  he  found  the  following  average  composition: 
C  43.69;  H  5.60;  N  16.87;  S  0.47;  P  5.23;  Ca  1.71  per  cent.  Where  the 
A-histon  is  to  be  found,  if  it  is  not  contained,  as  Malengreau  believes, 
in  the  nucleoproteids,  must  be  further  investigated. 

The  nucleohiston  prepared  by  Huiskamp  's  method  by  precipitating  with  CaCl2 
is,  according  to  him,  a  mixture  of  two  nucleohistons,  of  which  one,  the  a-nucleo- 
histon,  contains  4.5  per  cent  phosphorus  and  the  other,  /9-nucleohiston,  contains, 
on  the  contrary,  only  in  round  numbers,  3  per  cent  phosphorus.1  As  the  two 
nucleohistons  are  poorer  in  phosphorus  than  the  nucleic  acid-histon  compound 
analyzed  by  Bang,  and  as  Huiskamp  on  cleavage  of  his  preparation  did  not,  like 
Bang  and  .Malengreau,  obtain  pure  nucleic  acid,  it  is  still  a  question  whether 
JIi  iskamp  was  working  with  sufficiently  pure  substances. 

1  Zeitschr.  f.  physiol.  Chein.,  39. 


THE   THYMUS.  231 

In  regard  to  the  methods  used  by  the  above  investigators  in  the  isola- 
tion of  the  bodies  in  question  we  must  refer  to  the  original  publication-. 

In  connection  with  the  so-called  nucleohiston  attention  must  be  called  to  tissue 
fibrinogen  and  cell  fibrinogen,  which  are  compound  proteids,  and  claimed  by  certain 
investigators  to  stand  in  dose  relation  to  the  coagulation  of  the  blood.  These 
may  be  in  pari  nucleoproteids  and  in  pari  also  nucleohistons.    To  this  Bame  group 

belong  also  the  importanl  cell  constituents  described  by  Alex.  Schmidt1  and 
called  cytoglobin  and  pr&globulin.  The  cytoglobuiin,  which  is  soluble  in  water, 
may  he  considered  as  the  alkali  compound  of  priiglobulin.  The  residue  of  the 
cells  left  after  complete  extraction  with  alcohol,  water,  and  salt  solution  has  been 
called  cytin  by  Alex.  Schmidt. 

Besides  the  above-mentioned  and  the  ordinary  bodies  belonging  to  the 
connective- tissue  group,  small  quantities  of  fat,  leucin,  succinic  acid,  lactic 
acid,  sugar,  and  traces  of  iodothyrin  are  present.  According  to  Gautier  2 
ars<  nic  also  occurs  in  very  small  amounts,  and  no  doubt  here  as  well  as  in 
other  organs  it  is  related  to  the  nuclein  substances.  The  richness  in  nuclein 
bodies  explains  the  occurrence  of  large  quantities  of  purin  bases,  chiefly 
adenine,  whose  quantity,  according  to  Kossel  and  Schindler,3  is  1.79  p.  m. 
in  the  fresh  organ  and  19.19  p.  m.  in  the  dry  substance.  The  bodies  thymin 
and  uracil  (?)  obtained,  besides  lysin  and  ammonia  by  Kutscher,  as  prod- 
ucts of  autodigestion  of  the  gland,  probably  have  a  similar  origin,  although 
the  uracil  has  its  origin  from  histidin.  Lilienfeld  4  has  found  inosite  and 
protagon  in  the  cells  of  the  thymus.  The  quantitative  composition  of  the 
lymphocytes  of  the  thymus  of  a  calf  is,  according  to  Lilienfeld  's  analysis, 
as  follows.     The  results  are  given  in  1000  parts  of  the  dried  substance. 

Proteids 17.6 

Leuconuclein 687 . 9 

Histon 86 . 7 

Lecithin 75 . 1 

Fat 40.2 

Cholesterin 44 . 0 

Glycogen 8.0 

The  dried  substance  of  the  leucocytes  amounted  to  an  average  of  114.9 
p.  m.  Potassium  and  phosphoric  acid  are  prominent  mineral  constituents. 
Lilienfeld  found  KH2P04  amongst  the  bodies  soluble  in  alcohol. 

Attention  must  be  called  to  the  analyses  of  Banc5  which  show  that  the 
thymus  contains  about  the  same  quantity  of  nucleoproteid,  but  about  five 
times  as  much  histon  nucleate  as  the  lymphatic  glands — calculated  in  both 
cases  upon  the  same  amount  of  dry  substance.     Oidtmann  8  found  S07.06 

1  See  foot-note  5,  page  118. 
3Compt.  rend.,  129. 

3  Zeitschr.  f.  physiol.  Chem.,  13. 

4  Kutscher,  ibid.,  34;   Lilienfeld,  ibid.,  18. 

5  L.  c.  Arch.  f.  Math.,  etc. 

8  Cited  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem.,  4  Aufl.,  732. 


232  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

p.  m.  water,  192.74  p.  m.  organic,  and  0.2  p.  m.  inorganic  substances  in  the 
gland  of  a  child  two  weeks  old. 

The  Spleen.  The  pulp  of  the  spleen  cannot  be  freed  from  blood.  The 
mass  which  is  separated  from  the  spleen  capsule  and  the  structural  tissue 
by  pressure  and  which  ordinarily  serves  as  material  for  chemical  investiga- 
tions is  therefore  a  mixture  of  blood  and  spleen  constituents.  For  this 
reason  the  proteids  of  the  spleen  are  little  known.  As  characteristic  con- 
stituents there  are  albuminates  containing  iron,  and  especially  a  protein 
substance  which  does  not  coagulate  on  boiling,  and  which  is  precipitated 
by  acetic  acid  and  yields  an  ash  containing  much  phosphoric  acid  and  iron 
oxide.1 

The  pulp  of  the  spleen,  when  fresh,  has  an  alkaline  reaction,  but  quickly 
turns  acid,  due  partly  to  the  formation  of  free  paralactic  acid  and  partly 
perhaps  to  glycerophosphoric  acid.  Besides  these  two  acids  there  have- 
been  found  in  the  spleen  also  volatile  fatty  acids,  as  formic,  acetic,  and  butyric 
acids,  as  well  as  succinic  acid,  neutral  fats,  cholesterin,  traces  of  leucin,  inosite 
(in  ox-spleen),  scyllite,  a  body  related  to  inosite  (in  the  spleen  of  Plagiostoma), 
glycogen  (in  dog-spleen),  uric  acid,  xanthine  bodies,  and  jecorin.  Levene  2 
has  found  in  the  spleen  a  glucothionic  acid,  i.e.,  an  acid  which  is  related  to 
chondroitin-sulphuric  acid  but  not  identical  therewith,  and  which  gives  a 
beautiful  violet  coloration  with  orcin  and  hydrochloric  acid. 

Among  the  enzymes  occurring  in  the  spleen  the  most  important  is  the 
proteolytic  enzyme  first  detected  by  Hedin  and  Rowland,  and  also  occur- 
ring in  the  lymph-glands,  liver,  and  other  organs.  This  enzyme,  which  is 
most  active  in  acid  solutions,  not  only  acts  autolytically  upon  the  proteids 
of  the  spleen,  but  also  dissolves  fibrin.  According  to  the  more  recent 
investigations  of  Hedin  3  the  spleen  always  contains  two  proteolytic 
enzymes,  of  which  one  (lieno-a-protease)  acts  chiefly  in  alkaline  solution, 
while  the  other  (lieno-/?-protease)  is  only  active  in  acid  solution.  The 
/^-protease  goes  into  solution  on  extracting  the  spleen  with  0.2  per  cent 
acetic  acid,  while  the  a-protease  can  be  extracted  from  the  residue  by  a  5 
per  cent  NaCl  solution.  In  the  autolysis  of  the  spleen  Leathes  found 
proteoses,  lysin,  arginin,  histidin,  leucin,  aminovalerianic  acid,  aspartic 
acid,  and  tryptophan  among  the  cleavage  products.  Schumm  4  found,  in 
the  autolysis  of  a  leucsemic  spleen,  besides  leucin  and  tyrosin  relatively  large 
quantities  of  ammonia  and  lysin. 

Among  the  constituents  of  the  spleen  the  deposit  rich  in  iron,  which 
consists  of  ferruginous  granules  or  conglomerate  masses  of  them  and  which  are 

1  Cited  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem.,  4  Aufl.,  717. 

2  Levene,  Zeitschr.  f.  physiol.  Chem.,  37. 

3  Joarn.  of  Physiol. ,  30. 

4  Hedin  and  Rowland,  Zeitschr.  f.  physiol.  Chem.,  32j  Leathes,  Journ.  of  Physiol.^ 
28;  Schumm,  Hofmeister's  Beitriige,  3. 


THE  SPLEEN.  233 

derived  from  a  transformation  of  the  red  blood-corpuscles,  and  closely- 
studied  by  Nasse,  is  of  special  interest.  This  deposit  does  not  occur  to 
the  same  extent  in  the  spleen  of  all  animals.  It  is  found  especially  abun- 
dant in  the  spleen  of  thehoise.  Nasse  '  on  analyzing  the  grains  (from  the 
spleen  of  a  horse)  obtained  840-630  p.  m.  organic  and  160-370  p.  m.  inor- 
ganic substances.  These  last  consisted  of  500-720  p.  m.  Fe203,  205-388 
p.  m.  P._,( )-.  and  57  p.  m.  earths.  The  organic  substances  consisted  chiefly 
of  proteids  (000-800  p.  m.),  nuclein,  52  p.  m.  (maximum),  a  yellow  color- 
ing matter,  extractive  bodies,  fat,  eholesterin,  and  lecithin. 

In  regard  to  the  mineral  constituents  it  is  to  be  observed  that  the  amount 
of  sodium  and  phosphoric  acid  is  smaller  than  that  of  potassium  and  chlo- 
rine. The  amount  of  iron  in  new-born  and  young  animals  is  small  (La- 
picque,  Kruger,  and  Pernou),  in  adults  more  appreciable,  and  in  old  ani- 
mals sometimes  very  considerable.  Nasse  found  nearly  50  p.  m.  iron  in  the 
dried  pulp  of  the  spleen  of  an  old  horse.  Guillemonat  and  Lapicqtje  : 
have  determined  the  iron  in  man.  They  find  no  regular  increase  with 
growth,  but  in  most  cases  0.17-0.39  p.  m.  (after  subtracting  the  blood-iron) 
calculated  on  the  fresh  substance.  A  remarkably  high  amount  of  iron  is 
not  dependent  upon  old  age,  but  is  a  residue  from  chronic  diseases. 

The  quantitative  analyses  of  the  human  spleen  by  Oidtmaxx  3  give  the 
following  results:  In  men  he  found  750-094  p.  m.  water  and  250-306  p.  m. 
solids.  In  that  of  a  woman  he  found  774.8  p.  m.  water  and  225.2  p.  m. 
solids.  The  quantity  of  inorganic  bodies  was  in  men  4.9-7.4  p.  m.,  and  in 
women  9.5  p.  m. 

In  regard  to  the  pathological  processes  going  on  in  the  spleen  we  must 
specially  recall  the  abundant  re-formation  of  leucocytes  in  leucaemia  and 
the  appearance  of  amyloid  substance  (see  page  54) . 

The  physiological  functions  of  the  spleen  are  little  known,  with  the 
exception  of  its  importance  in  the  formation  of  leucocytes.  Some  consider 
the  spleen  as  an  organ  for  the  dissolution  of  the  red  blood-corpuscles,  and 
the  occurrence  of  the  above-mentioned  deposit  rich  in  iron  seems  to  con- 
firm this  view.  The  spleen  has  also  been  claimed  to  play  a  certain  part  in 
digestion.  This  organ  is  said  by  Schiff,  Herzen,  Gachet  and  Pachon 
to  be  of  importance  in  the  production  of  trypsin  in  the  pancreas.  The 
investigations  of  Herzex  seem  to  confirm  this  relation,  although  it  is  still 
difficult  to  give  an  opinion  on  the  intricate  question  (see  also  Heidexhaix, 
Ewald  *) . 

1  Maly's  Jahresber.,  19,  315. 

:  I.apicque,  ibid.,  20;  Lapicque  and  Guillemonat,  Compt.  rend,  de  Soc.  biol.,  4S, 
and  Arch,  de  Physiol.  (5),  S;  Kruger  and  Pernou,  Zeitschr.  f.  Biologie,  27;  Xasse, 
cited  from  Hoppe-Seyler,  Physiol.  Chem.,  720. 

5  Cited  from  v.  Gorup-Besanez,  Lehrbuch,  4  Aufl.,  719. 

4  Schiff.  cited  by  Herzen,  Pfluger's  Arch.,  30,  295  and  308,  and  Maly's  Jahresber., 
18j  Gachet  and  Pachon,  Arch,  de  Physiol.  (5),  10;  Heidenhain  in  Herrmann's  Handb. 


234  CHYLE.  LYMPH.  TRANSUDATES   AXD  EXUDATES. 

An  increase  in  the  quantity  of  uric  acid  eliminated  has  been  observed 
by  many  investigators  (see  Chapter  XT)  in  lineal  leucaemia,  while  the 
reverse  has  been  observed  under  the  influence  of  quinine  in  large  doses, 
which  produces  an  enlargement  of  the  spleen.  These  facts  give  a  rather 
positive  proof  that  there  is  a  close  relationship  between  the  spleen  and 
the  formation  of  uric  acid.  This  relationship  has  been  studied  by  Horbac- 
-:n.  He  has  shown  that  when  the  spleen-pulp  and  blood  of  calves 
are  allowed  to  act  on  each  other,  under  certain  conditions  and  temperature, 
in  the  presence  of  air,  large  quantities  of  uric  acid  are  formed.  Under 
other  conditions  he  obtained  from  the  spleen-pulp  only  xanthine  bodies 
with  very  little  or  no  uric  acid.  Horbaczewski  1  has  also  shown  that  the 
uric  acid  originates  from  the  nucleins  of  the  spleen,  which  yield  uric  acid 
and  xanthine  bodies  according  to  the  experimental  conditions.  A  connec- 
tion between  the  spleen  and  uric-acid  formation  is  a  priori  to  be  expected 
on  account  of  the  large  quantities  of  nuclein  contained  in  this  organ;  but 
that  the  spleen  has  a  special  relation  to  the  uric-acid  formation  as  com- 
pared to  other  organs  rich  in  nuclein  has  not  been  proven,  nor  is  it  prob- 
able (see  Chapter  XV). 

The  spleen  has  the  same  property  as  the  liver  of  retaining  foreign  bodies, 
metals  and  metalloids. 

The  Thyroid  Gland.  The  nature  of  the  different  protein  substances 
occurring  in  the  thyroid  gland  has  not  been  sufficiently  studied,  but  at 
rrt.  through  the  researches  of  Oswald,  there  are  known  at  least  two 
bodies  which  are  constituents  of  the  so-called  secretion  of  the  glands.  One 
of  these,  iodothyreogtobulin.  behaves  like  a  globulin,  while  the  other  is  a 
nucleoproteid  (see  also  Gourlat  2).  The  iodine  present  in  the  gland 
occurs  chiefly  in  the  first  body,  while  the  arsenic,  which  has  been  shown  to  be 
a  normal  constituent  by  Gautier  and  Bertrand,3  seems  to  be  related  to 
the  nuclein  substance- . 

According  to  Oswald  the  iodottryreoglobulin  only  occurs  in  those 
glands  which  contain  colloid,  while  the  colloid-free  glands,  the  parenchyma- 
goitre,  and  the  glands  of  the  new-born  contain  thyreoglobulin  free 
from  iodine.  The  thyreoglobulin  first  becomes  iodized  into  iodothyreo- 
globulin  on  passing  from  the  follicle  cells.  Besides  these  mentioned  bodies 
-  .  xartihin-e.  hypoxanthim.  iodothynn,  lactic  and  succinic  acids  occur 
in  the  thyreoidea.   Oidtmann  4  found  in  the  thyroid  gland  of  an  old  woman 

d.  PL-  5    Absonderungsvorgange,  206;    Ewald,  Verhandl.  d.  physiol.  Gesellsch. 

in  Berlin.  "  •  ~  -       -    -  aUo  Chapter  IX. 

natshefte  f.  Chem.,  10,  and  "MVien.  Sitzungsber.  Math.  Nat.  Klasse,  100,  Abth.  3. 
irlay,  Journ.  of  Physiol.,  16;    Oswald,  Zeitschr.  f.  physiol.  Chem.,  32,  and 
Biochera.  Centralbl.,  1,  2-19. 

2  Gautier,  Corapt.  rend.,  129.  See  also  ibid.,  130,  131,  134,  135;  Bertrand,  ibid., 
1*4,  I  * 

4  L.  c,  732. 


THE   THYROID  GLAND. 

822.4  p.  in.  water,  176.6  p.  m.  organic  and  0.9  p.  m.  inorganic  substances. 
He  found  772.1  p.  in.  water.  223.5  p.  m.  organic  and  4.4  p.  m.  inorganic 
substances  in  an  infant  two  weeks  old. 

In  "struma,  cystica"  Hoppe-Seyler  found  hardly  any  prote.d  in  the  smaller 
glandular  vessels,  but  an  excess  of  mucin,  while  in  the  larger  he  found  a  great 
deal  of  prottidf  70-80  p.  m.1  Cholesierin  is  regularly  found  in  such  cysts,  some- 
times in  such  large  quantities  that  the  entire  contents  form  a  thick  mass  of  cho- 
lesterin  plates.  Crystals  of  calcium  oxalate  also  occur  frequently.  The  contents 
of  the  struma  cysts  are  sometimes  of  a  brown  color  due  to  decomposed  coloring- 
matter,  mcth&moglobin  (and  hannatin?).  Bile-coloring  matters  have  also  been 
found  in  such  cysts.  (In  regard  to  the  paraJbttmiru  and  colloid*  which  have  been 
found  in  struma  cysts  and  colloid  degeneration,  see  Chapter  XIII.) 

Those  substances  which  bear  a  close  relationship  to  the  functions  of 
the  gland  seem  to  be  of  special  interest. 

The  complete  extirpation,  as  also  the  pathological  destruction,  of  the 
thyroid  gland  causes  great  disturbances,  ending  finally  in  death.  In  dogs, 
after  the  total  extirpation,  a  disturbance  of  the  nervous  and  museula: 
terns  occurs,  such  as  trembling  and  convulsions,  and  death  generally  super- 
venes shortly  after,  most  often  during  such  an  attack.2  In  human  beings 
different  disturbances  appear,  such  as  nervous  symptoms,  diminished  intel- 
ligence, dryness  of  the  skin,  falling  out  of  the  hair,  and,  on  the  whole,  those 
symptoms  which  are  included  under  the  name  cachexia  thyreopriva.  and 
death  follows  gradually.  Among  these  symptoms  must  be  mentioned  the 
peculiar  slimy  infiltration  and  exuberance  of  the  connective  tissue  called 
niyxcedema.  It  has  been  proved  that  the  destructive  action  of  the  removal 
of  the  thyroid  can  be  counteracted  by  the  artificial  introduction  of  extracts 
of  the  thyroid  gland  into  the  body,  and  even  by  feeding  with  the  substance 
of  the  gland.  On  the  other  hand,  it  has  been  observed  on  administering 
too  large  quantities  of  gland  substance  that  threatening  symptoms  and 
disturbances  occur  in  man  as  well  as  in  animals.  From  a  physiologico- 
chemical  standpoint  the  abnormally  increased  destruction  of  body  proteid, 
occurring  on  continuous  feeding  with  thyroid  preparations,  is  of  the 
greatest  importance. 

From  this  it  follows  that  the  glands  contain  specifically  active  substances. 
It  is  impossible  for  the  present  to  state.anything  about  the  importance  of 
the  bases  found  by  certain  investigators,  such  as  S.  Frankel,  Drechsel, 
and  Kocher;3  these  bodies  have  not  been  characterized  sufficiently.  It 
seems  positively  proven  that  the  specifically  active  substance  is.  in  greater 

1  Physiol.  Chem.,  721. 

2  The  divergent  statements  as  to  the  necessity  of  the  thyroid  gland  can  be  found  in 
H.  Munk,  Yirchow's  Arch.,  150. 

s  Frankel,  Wien.  med.  Blatter,  1S95  and  1896;  Drechsel  and  Kocher,  Centralbl. 
f.  Physiol.,  9,  705. 


236  CHYLE,  LYMPH,   TRANSUDATES,   AND  EXUDATES. 

part,  if  not  entirely,  as  first  shown  by  Notkin,1  a  protein  substance: 
Notkin's  thyreoproteid,  Oswald's  thyreoglobulin.  This  does  not  con- 
tradict the  views  of  Baumann  and  Roos  that  the  active  substance  is  iodo- 
thyrin, as  this  is  produced  as  a  cleavage  product  from  the  iodothyreo- 
globulin. 

Iodothyrin  is  considered  by  Baumann,  who  first  showed  that  the  thyroid  con- 
tained iodine  and  who  with  Roos  2  showed  the  importance  of  this  substance  for  the 
physiological  activity  of  the  gland,  as  the  only  active  substance.  Iodothyrin  was 
obtained  by  Baumann  by  boiling  the  finely  divided  gland  with  dilute  sulphuric 
acid  as  an  amorphous,  brown  mass  nearly  insoluble  in  water  but  readily  soluble 
in  alkali  and  precipitated  again  by  the  addition  of  acid.  The  iodothyrin, 
which  is  not  a  unit  body,  has  a  variable  content  of  iodine  and  is  not  a  protein 
substance. 

Thyreoglobulin  was  obtained  by  Oswald  from  the  watery  extract  of 
the  gland  by  half  saturating  with  ammonium  sulphate.  It  has  the  proper- 
ties of  the  globulins  and  with  the  exception  of  the  iodine  content  it  has 
about  the  same  composition  as  the  proteids.  The  amount  of  iodine  varies": 
0.46  per  cent  in  pigs,  0.86  per  cent  in  oxen,  and  0.34  per  cent  in  man.  In 
young  animals,  whose  glands  contain  no  iodine,  the  thyreoglobulin  is 
iodine-free.  Thyreoglobulin  on  taking  up  iodine  is  converted  into  iodo- 
thyreoglobulin.  By  introducing  iodine  salts  the  iodine  content  of  the 
iodothyreoglobulin  can  be  raised  in  living  animals  and  thereby  also  the 
physiological  activity  increased  (Oswald).  The  amount  of  iodine  in  the 
gland  is  markedly  dependent  upon  the  food. 

According  to  Oswald  iodothyreoglobulin,  as  a  physiological  excitant 
upon  the  nervous  system,  has  a  regulating  action  upon  metabolism. 
The  exclusion  of  this  action,  after  destruction  or  extirpation  of  the  gland, 
explains,  according  to  Oswald,  the  injurious  results  produced  by  these 
changes  upon  the  gland.  According  to  Blum  the  thyroid  gland  removes 
from  the  blood  a  poisonous  body,  the  thyreotoxalbumin,  and  makes  it  non- 
injurious  by  taking  up  iodine.  We  cannot  enter  further  into  this  and  other 
related  questions. 

The  Suprarenal  Capsule.  Besides  proteids,  substances  of  the  connective 
tissue,  and  salts,  there  occur  in  the  suprarenal  capsule  inosite,  purin  bases,  espe- 


xWien.  med.  Wochenschr. ,  1895,  and  Virchow's  Arch.,  144,  Suppl.,  224. 

2  In  regard  to  this  subject,  see  Baumann  and  Roos,  Zeitschr.  f.  physiol.  Chem.,  21 
and  22;  also  Baumann,  Munch,  med.  Wochenschr.,  1896;  Baumann  and  Goldmann, 
ibid. ;  Roos,  ibid.  An  extensive  review  of  the  literature  on  the  action  of  iodothyrin 
and  the  thyroid  preparations  can  be  found  in  Roos,  Zeitschr.  f.  physiol.  Chem.,  22,  18. 
In  regard  to  their  action  in  proteid  destruction  and  metabolism  see  F.  Voit,  Zeitschr. 
f.  Biologie,  35;  Schondorff,  Pfliiger's  Arch.,  67,  and  Anderson  and  Bergman,  Skand. 
Arch.  f.  Physiol.,  8.  A  summary  of  the  thyroid  literature  for  the  last  years  is  found  in 
Maly's  Jahresber. ,  24  and  25.  See  also  the  works  of  Blum  and  Oswald,  cited  by  Oswald 
in  Biochem.  Centralbl.,  1,  249. 


SUPRARENAL  CAPSULE.  237 

cially  xanthine  (Oker-Blom1),  relatively  considerable  lecithin  and  neurin, 
and  glycero phosphoric  acid,  which  are  probably  decomposition  products 
of  the  lecithin.  The  older  statements  on  the  occurrence  of  benzoic 
acid,  hippuric  acid,  and  bile-acids  are,  on  the  contrary,  doubtful  and  are 
not  substantiated  by  recent  investigations  (Stadelmann).  In  the  medulla 
older  investigators,  like  Vulpian  and  Arnold,  found  a  chromogen  which 
was  considered  to  be  connected  with  the  abnormal  pigmentation  of  the 
skin  in  Addison's  disease.  This  chromogen  which  is  transformed  by  air, 
light,  alkalies,  iodine,  and  other  bodies  into  a  red  pigment,  seems,  on  the 
contrary,  to  be  related  to  the  substance  of  the  gland  producing  an  increase 
in  the  blood-pressure. 

Adrenalin  (suprarenin,  epinephrin).  That  the  watery  extract  of  the 
suprarenal  capsule  has  a  blood-pressure-raising  action  was  shown  by  Oliver 
and  Schafer,  Cybulski  and  Szymonowicz.2  The  substance  which  is  here 
active  was  formerly  called  sphygmogenin,  but  has  recently  been  investi- 
gated by  several  experimenters,  especially  v.  Furth,  Abel,  Takamixe,  and 
Aldrich,3  and  is  now  called  suprarenin  (v.  Furth),  epinephrin  (Abel), 
adrenalin  (Takamixe).  Adrenalin  is  soluble  in  water,  precipitable  by 
ammonia  as  a  crystalline  body,  and  on  account  of  its  changeability  exact 
investigations  have  been  made  with  difficulty.  According  to  v.  Firth, 
Aldrich 's  formula,  C9H13N03,  is  correct.  It  is  a  cyclic  compound  which, 
according  to  v.  Furth,  contains  three  hydroxyl  groups  and  one  methylamine 
group  and  for  which  he  considers  the  formula  [(CH3)NC2H(OH)]C6H6(OH)2 
as  correct.  Adrenalin  yields  pyrrol  and  skatol  and  gives  protocatechuic 
acid  as  cleavage  product  with  alkali. 

Adrenalin  gives  an  emerald-green  reaction  with  ferric  chloride  in  acid 
solution  and  a  crimson  one  in  alkaline  solution.  It  reduces  Fehling's  solu- 
tion and  an  ammoniacal  silver  solution.  Epinephrin  (Abel)  is  precipitated 
by  several  alkaloid  reagents  and  gives  color  reactions  with  Mandelin's  alka- 
loid reagent  and  with  permanganate  and  sulphuric  acid.  On  this  point  the 
conditions  are  not  quite  clear.  According  to  Abel,  who  gives  the  formula 
C10H13XO3  to  his  epinephrin,  the  crystalline  substance  C10H13NO3+ '.H,0 
(epinephrin  hydrate)  precipitated  by  ammonia,  which  corresponds  to  the 
adrenalin  of  the  other  investigators,  does  not  have  the  alkaloid  properties 

1  Oker-Blom,  Zeitschr.  f.  physiol.  Chem.,  28;  Stadelmann,  ibid.,  18,  which  also 
contains  the  literature  on  this  subject. 

1  Oliver  and  Schafer,  Proceed,  of  Physiol.  Soc.  London,  1895.  Further  literature 
on  the  function  of  the  suprarenal  capsule  may  be  found  in  Szymonowicz,  Pfluger's 
Arch.,  64. 

3  The  literature  here  necessary  may  be  found  in  v.  Furth,  Zeitschr.  f.  physiol.  Chem., 
23,  26,  29,  and  Wien.  Sitzungsber.  Math.  Nat.  KX,  112,  1903.  See  also  Abel,  Zeitschr. 
f.  physiol.  Chem.,  28;  Amer.  Journ.  of  Physiol.,  1899,  and  The  Johns  Hopkins  Hospi- 
tal Bull.,  No.  76  (1897),  90  and  91  (1898),  120  and  128  (1901),  131  and  132  (1902); 
Ber.  d.  d.  chem.  Gesellsch.,  36. 


238  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

of  epinephrin,  but  the  compound  obtains  them  by  the  action  of  mineral  acids 
and  is  then  converted  into  epinephrin.  Further  investigation  is  necessary 
before  this  can  be  explained. 

The  glycosuria  first  observed  by  Blum  after  the  injection  of  the  extract 
of  the  suprarenal  capsule  is  due  to  an  action  of  the  adrenalin,  and  it  is  hardly 
possible  that  the  diastatic  enzyme  found  in  the  suprarenal  capsule  by  Crof- 
tan  *  takes  any  part  in  this  change. 

1  Blum,  Pfluger's  Arch.,  90j  Croftan,  ibid.,  90. 


CHAPTER  VIII. 
THE  LIVER. 

The  liver,  which  is  the  largest  gland  of  the  body,  stands  in  close  rela- 
tionship to  the  blood-forming  glands.  The  importance  of  this  organ  for 
the  physiological  composition  of  the  blood  is  evident  from  the  fact  that  the 
blood  coming  from  the  digestive  tract,  laden  with  absorbed  bodies,  must 
circulate  through  the  liver  before  it  is  driven  by  the  heart  through  the 
different  organs  and  tissues.  It  has  been  proved,  at  least  for  the  carbo- 
hydrates, that  an  assimilation  of  the  absorbed  nutritive  substances  which  are 
brought  to  the  liver  by  the  blood  of  the  portal  vein  takes  place  in  this  organ, 
and  there  is  no  doubt  that  synthetical  processes  also  occur.  The  occurrence 
of  synthetical  processes  in  the  liver  has  been  positively  proved  by  special 
observations.  It  is  possible  that  in  the  liver  certain  ammonia  combinations 
are  converted  into  urea  or  uric  acid  (in  birds)  (see  Chapter  XV) ,  while  certain 
products  of  putrefaction  in  the  intestine,  such  as  phenols,  may  be  con- 
verted by  synthesis  into  ethereal  sulphuric  acids  by  the  liver  (Pfluger  and 
Kochs,  Embden  and  Glaessner),  probably  also  converted  into  conju- 
gated glucuronic  acids  (Embden  x).  The  liver  has  also  the  property  of 
removing  and  retaining  heterogeneous  bodies  from  the  blood,  and  this  is 
not  only  true  of  metallic  salts,  which  are  often  removed  by  this  organ,  but 
also,  as  Schipf,  Lautenberger,  Jacques,  Heger,  and  especially  Roger 
have  shown,  the  alkaloids  are  retained,  and  are  probably  also  partially 
decomposed  in  the  liver.  Toxins  are  also  withheld  by  the  liver,  and  hence 
this  organ  has  a  protective  action  against  poisons.2  The  researches  of 
Bouchard,  Roger  and  Mairet  and  Vires  3  has  shown  that  the  liver  itself 
may  have  a  poisonous  action. 

Even  though  the  liver  is  of  assimilatory  importance  and  purines  the 
blood  coming  from  the  digestive  tract,  it  is  at  the  same  time  a  secretory 
organ  which  eliminates  a  specific  secretion,  the  bile,  in  the  production  of 

1  Pfluger  and  Kochs,  Pfluger 's  Arch.,  20  and  24;  Embden  and  Glaessner,  Hof- 
meister's  Beitriige,  1;   Embden,  ibid.,  2. 

2  Roger,  Action  du  foie  sur  les  poisons  (Paris,  1887),  which  also  contains  the  older 
literature;  Bouchard,  Lecons,  sur  les  autointoxications  dans  les  Maladies  (Paris,  1887) ; 
and  E.  Kotliar  in  Arch,  des  sciences  biologique  de  St.  P6tersbourg,  2. 

3  See  Mairet  and  Vires,  Arch,  de  Physiol.  (5),  9. 

239 


240  THE  LIVER. 

which  the  red  blood-corpuscles  are  destroyed,  or  at  least  one  of  their  con- 
stituents, the  haemoglobin.  It  is  generally  admitted  that  the  liver  acts 
contrariwise  during  fcetal  life,  at  that  time  forming  the  red  blood-cor- 
puscles. 

There  is  no  doubt  that  the  chemical  operations  going  on  in  this  organ 
are  manifold  and  must  be  of  the  greatest  importance  for  the  organism;  but 
unfortunately  very  little  is  known  about  the  kind  and  extent  of  these  pro- 
cesses. Our  knowledge  on  this  subject  has  been  essentially  advanced  by 
the  recent  investigations  on  the  enzymes  of  the  liver,  as  well  as  by  the 
autolytic  processes  in  this  organ,1  but  even  here  it  must  be  admitted  that 
our  knowledge  of  the  character  and  extent  of  these  changes  is  small.  Among 
the  products  of  these  chemical  processes  there  are  two  which  are  especially 
important  and  must  be  treated  in  this  chapter,  namely,  the  glycogen  and 
the  bile.  Before  the  study  of  these  products  is  taken  up  a  short  discus- 
sion of  the  constituents  and  the  chemical  composition  of  the  liver  is  neces- 
sary. 

The  reaction  of  the  liver-cells  is  alkaline  towards  litmus  during  life, 
but  becomes  acid  after  death,  due  to  a  formation  of  lactic  acid,  chiefly 
fermentation  lactic  acid  and  other  organic  acids  (Morishima,  Magnus- 
Levy  2) .  A  coagulation  of  the  protoplasmic  proteids  in  the  cells  probably 
takes  place.  A  positive  difference  between  the  proteids  of  the  dead  and 
the  living,  non-coagulated  protoplasm  has  not  been  observed. 

The  proteids  of  the  liver  were  first  carefully  investigated  by  Plosz.  He 
found  in  the  watery  extract  of  the  liver  an  albuminous  substance  which 
coagulates  at  45°  C,  also  a  globulin  which  coagulates  at  75°  C,  a 
nucleoalbumin  which  coagulates  at  70°  C,  and  lastly  a  proteid  body 
which  is  nearly  related  to  the  coagulated  albumins  and  which  is  insoluble  in 
dilute  acids  or  alkalies  at  the  ordinary  temperature,  but  dissolves  on  the 
application  of  heat,  being  converted  into  an  albuminate.  Halliburton3 
has  found  two  globulins  in  the  liver-cells,  one  of  which  coagulates  at 
68-70°  C,  and  the  other  at  45-50°  C.  He  also  found,  besides  traces  of 
albumin,  a  nucleoproteid  which  possessed  1.45  per  cent  phosphorus  and  a 
coagulation- point  of  60°  C.  Besides  these  proteids,  the  liver-cells  con- 
tain a  large  quantity  of  a  difficultly  soluble  protein  substance  (see  Plosz). 
It  also  contains,  as  first  shown  by  St.  Zaleski  and  then  substantiated  by 
several  other  investigators,  ferruginous  proteids  of  different  kinds.4  The 
chief  portion  of  the  protein  substances  in  the  liver  seems  to  consist  in  fact 

1  See  especially  the  works  of  Jacoby,  Zeitschr.  f.  physiol.  Chem.,  30;  Conradi,  Hof- 
meister's  Beitrage,  1;  Magnus-Levy,  ibid.,  2. 

2  Morishima,  Arch.  f.  exp.  Path.  u.  Pharm.,  43;  Magnus-Levy,  1.  c. 
'Plosz,  Pfliiger's  Arch.,  7;  Halliburton,  Journ.  of  Physiol.,  13,  Suppl.,  1892. 

*  St.  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  10,  486]  Woltering,  ibid.,  21:  Spitzer, 
Pfliiger's  Arch.,  67. 


COMPOSITION  OF   Tin:   LIVER.  241 

of  ferruginous  nucleoproteids.  On  boiling  the  liver  with  water,  such  a 
nucleoproteid  or  perhaps  several  are  split,  and  a  solution  Is  obtained  contain- 
ing a  nucleic-acid-rich  nucleoproteid  or  a  mixture  of  these  which  are  pre- 
cipitable  by  acids.  This  proteid  or  proteid  mixture  has  been  called  ferra- 
tin  by  Schmiedeberg,1  and  this  yields  on  splitting  with  acids,  besides 
nuclein  bases,2  also  a  pentose  which  Wohlgemuth3  has  shown  to  be  1-xylose. 

The  yellow  or  brown  pigment  of  the  liver  has  been  little  studied.  Dastrb 
and  Floresco  4  differentiate  in  vertebrates  and  certain  invertebrates  between  a 
ferruginous  pigment  soluble  in  water,  ferrine,  and  a  pigment  soluble  in  chloro- 
form and  insoluble  in  water,  chlohochho.me.  They  have  not  isolated  these 
pigments  in  a  pure  condition.  In  certain  invertebrates  chlorophyll  originating 
from  the  food  also  occurs  in  the  liver. 

The  fat  of  the  liver  occurs  partly  as  very  small  globules  and  partly 
(especially  in  nursing  children  and  sucking  animals,  as  also  after  food  rich 
in  fat)  as  rather  large  fat-drops.  The  occurrence  of  a  fatty  infiltration,  i.e., 
a  transportation  of  fat  to  the  liver,  may  not  only  be  produced  by  an  excess 
of  fat  in  the  food  (Noel-Paton)  ,  but  also  by  a  migration  from  other  parts 
of  the  body  under  abnormal  conditions,  such  as  poisoning  with  phosphorus, 
phlorhizin,  and  certain  other  bodies  (Leo,  Rosenfeld,  and  others5).  If 
the  amount  of  fat  in  the  liver  is  increased  by  an  infiltration,  the  water 
decreases  correspondingly,  while  the  quantity  of  the  other  solids  remains 
little  changed.  Changes  of  such  a  kind  may  occur,  so  that,  because  of  the 
opposition  (Rosenfeld)  existing  between  glycogen  and  fat,  a  liver  rich 
in  fat  is  habitually  poor  in  glycogen.  The  reverse  occurs  after  feeding 
with  carbohydrate-rich  food,  namely,  the  liver  is  rich  in  glycogen  and 
poor  in  fat, 

The  composition  of  the  liver-fat  not  only  seems  to  vary  in  different 
animals,  but  is  variable  'with  changing  conditions.  Thus  Noel-Paton 
found  that  the  liver-fat  in  man  and  several  animals  was  poorer  in  oleic  acid 
and  had  a  correspondingly  higher  melting-point  than  the  fat  from  the 
subcutaneous  connective  tissue,  while  Rosenfeld  6  has  observed  the  opposite 
condition  on  feeding  dogs  with  mutton-fat. 

1  Arch.  f.  exp.  Path.  u.  Pharm.,  33;  see  also  Vay,  Zeitschr.  f.  physiol.  Chem.,  20. 

2  See  Beccari,  Arch,  italiennes  de  Biologie,  38. 

5  See  Salkowski,  Berl.  klin.  Wochenschr.,  1S95;  Hammarsten,  Zeitschr.  f.  physiol. 
Chem.,  19;  Blumenthal,  Zeitschr.  f.  klin.  Med.,  34;  Wohlgemuth,  Berl.  klin.  Woch- 
enschr., 1900;  and  Zeitschr.  f.  physiol.  Chem.,  37. 

4  Arch,  de  Physiol.  (5),  10. 

'Noel-Paton,  Journ.  of  Physiol.,  19;  Leo,  Zeitschr.  f.  physiol.  Chem.,  9;  Athan- 
asiu,  Pfliiger's  Arch.,  74;  Taylor,  Journ.  of  Exp.  Med.,  4;  Kraus  u.  Sommer,  Hofmeis- 
ter's  Beitriige,  2;  Rosenfeld,  Zeitschr.  f.  klin.  Med.,  36.  See  also  Rosenfeld,  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  I. 

*  Cited  by  Lummert,  Pfliiger's  Arch.,  71.  In  regard  to  the  liver-fat  of  children,  see 
Thiemich,  Zeitschr.  f.  physiol.  Chem.,  20. 


242  THE  LIVER. 

Lecithin  is  a  normal  constituent  of  the  liver,  and  amounts  to  about  23.5 
p.  m.  according  to  Noel-Paton.1  In  starvation  the  lecithin,  according  to 
Noel-Paton,  forms  the  greatest  part  of  the  ethereal  extract,  while  with 
food  rich  in  fat  it,  on  the  contrary,  forms  the  smallest  part.  Cholesterin 
only  occurs  in  small  quantities.  The  ethereal  extract  also  contains  a 
protagon-like  body,  jecorin. 

Jecorin  was  first  found  by  Drechsel  in  the  liver  of  horses,  and  also  in  the 
liver  of  a  dolphin,  and  later  by  Baldi  in  the  liver  and  spleen  of  other  animals,  in 
the  muscles  and  blood  of  the  horse,  and  in  the  human  brain.  It  contains  sul- 
phur and  phosphorus,  but  its  constitution  is  not  positively  known.  Jecorin  dis- 
solves in  ether,  but  is  precipitated  from  this  solution  by  alcohol.  It  reduces  copper 
oxide,  and  it  solidifies  after  boiling  with  alkalies  to  a  gelatinous  mass.  Manasse 
has  detected  dextrose  as  osazone  in  the  carbohydrate  complex  of  jecorin.  It 
may  lead  to  errors  in  the  investigations  of  organs  or  tissues,  for  it  can  easily  be 
mistaken  for  lecithin  on  account  of  its  solubilities  and  because  it  contains- 
phosphorus. 

The  statement  byBiNG2  that  jecorin  is  a  combination  of  lecithin  and  dextrose 
does  not  follow  from  the  analyses  of  jecorin  thus  far  known.  Jecorin  contains 
sulphur,  even  as  much  as  2.75  per  cent,  and  also  the  relation  of  P:N  in  lecithin  is 
1:1,  while  in  jecorin  it  is  about  1 :4. 

Among  the  extractive  substances  besides  glycogen,  which  will  be  treated 
later,  rather  large  quantities  of  the  xanthine  bodies  occur.  Kossel3  found 
in  1000  parts  of  the  dried  substance  1.97  p.  m.  guanine,  1.34  p.  m.  hypo- 
xanthine,  and  1.21  p.  m.  xanthine.  Adenine  is  also  contained  in  the  liver.  In 
addition  there  have  been  found  urea  and  uric  acid  (especially  in  birds), 
and  indeed  in  larger  quantities  than  in  the  blood,  paralactic  acid,  leucin, 
and  cystin.  In  pathological  cases  inosite  and  tyrosin  have  been  detected. 
The  occurrence  of  bile  coloring-matters  in  the  liver-cell  under  normal  con- 
ditions is  doubtful;  but  in  retention  of  the  bile  the  cells  may  absorb  the 
coloring-matter  and  become  colored  thereby. 

The  mineral  bodies  of  the  liver  consist  of  phosphoric  acid,  potassium, 
sodium,  alkaline  earths,  and  chlorine.  The  potassium  is  in  excess  of  the 
sodium.  Iron  is  a  regular  constituent  of  the  liver,  but  it  occurs  in  very 
variable  amounts.  Bunge  has  found  0.01-0.355  p.  m.  iron  in  the  blood- 
free  liver  of  young  cats  and  dogs.  This  was  calculated  on  the  liver  sub- 
stance freshly  washed  with  a  1  per  cent  NaCl  solution.  Calculated  on  10 
kilos  bodily  weight,  the  iron  in  the  liver  amounted  to  3.4-80.1  mg. 
Recent  determinations  of  the  quantity  of  iron  in  the  liver  of  the  rabbit, 
dog,  hedgehog,  pig,  and  man  have  been  made  by  Guillemonat  and  La- 
picque.     The  variation  was  great  in  human  beings.     In  men  the  quantity  of 

1  L.  c.     See  also  Hefter,  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 

2  Drechsel,  Ber.  d.  sachs.  Gesellsch.  d.  Wissensch. ,  1886,  44,  and  Zeitschr.  f.  Biol- 
ogie,  33;  Baldi,  Du  Bois-Reymond's  Arch.,  1887,  Suppl.,  100;  Manasse,  Zeitschr.  f. 
physiol.  Chem.,  20;   Bing,  Centralbl.  f.  Physiol.,  12,  and  Skand.  Arch.  f.  Physiol.,  9. 

3  Zeitschr.  f.  physiol.  Chem.,  8. 


IRON  IN  THE  LIVER.  243 

iron  in  the  blood-free  liver  (blood-pigment  subtracted  in  the  calculation) 
was  regularly  more,  and  in  women  less,  than  0.20  p.  m.  (calculated  on  the 
fresh  moist  organ).  Above  0.5  p.  111.  is  considered  as  pathological.  Accord- 
ing to  BlELFELD,1  who  also  finds  a  greater  iron  content  in  men,  this  differ- 
ence appears  only  after  the  first  20-25  years.  At  this  age  (20-25  years) 
the  iron  content  is  smallest. 

The  quantity  of  iron  in  the  liver  can  be  increased  by  drugs  containing 
iron,  as  also  by  inorganic  iron  salts,  and  the  largest  deposition  of  iron  was 
observed  by  Novi  2  after  the  hypodermic  injection  of  iron.  The  quantity 
of  iron  may  also  be  increased  by  an  abundant  destruction  of  red  blood- 
corpuscles,  which  will  result  from  the  injection  of  dissolved  haemoglobin  in 
which  process  the  iron  combinations  derived  from  the  blood-pigments  in 
other  organs,  such  as  the  spleen  and  marrow,  also  seem  to  take  part.1  A 
destruction  of  blood-pigments,  with  a  splitting  off  of  combinations  rich 
in  iron,  seems  to  take  place  in  the  liver  in  the  formation  of  the  bile- 
pigments.  Even  in  invertebrates,  which  have  no  haemoglobin,  the  so- 
called  liver  is  rich  in  iron,  from  which  Dastre  and  Floresco  4  conclude 
that  the  quantity  of  iron  in  the  liver  of  invertebrates  is  entirely  inde- 
pendent of  the  decomposition  of  the  blood-pigment,  and  in  vertebrates 
it  is  in  part  so.  According  to  these  authors  the  liver  has,  on  account  of 
the  quantity  of  iron,  a  specially  important  oxidizing  function,  which  they 
call  the  "  fondion  mod/ale"  of  the  liver. 

The  richness  of  the  liver  of  new-born  animals  in  iron  is  of  special  inter- 
est; a  condition  which  follows  from  the  analyses  of  St.  Zaleski,  but  was 
especially  studied  by  Kruger,  Meyer,  and  Pernou.  In  oxen  and  cows 
they  found  0.246-0.276  p.  m.  iron  (calculated  on  the  dry  substance),  and  in 
the  cow' -foetus  about  ten  times  as  much.  The  liver-cells  of  a  calf  a  week 
old  contain  about  seven  times  as  much  iron  as  the  adult  animal;  the 
quantity  sinks  in  the  first  four  weeks  of  life,  when  it  reaches  about  the 
same  amount  as  in  the  adult.  Lapicque5  has  also  found  that  in 
rabbits  the  quantity  of  iron  in  the  liver  steadily  diminishes  from  the  eighth 
day  to  three  months  after  birth,  namely,  from  10  to  0.4  p.  m.,  calculated 
on  the  dry  substance.  "The  foetal  liver-cells  bring  an  abundance  of  iron 
into  the  world  to  be  used  up,  within  a  certain  time,  for  a  purpose  not  well 
known.''  A  part  of  the  iron  exists  as  phosphate,  and  the  greater  part  in 
combination  in  the  ferruginous  protein  bodies  (St.  Zaleski). 

1  Bunge,  Zeitschr.  f.  physiol.  Chem.,  17,  78;  Guillemonat  and  Lapicque,  Compt. 
rend,  de  Soc.  biol.,  48,  and  Arch,  de  Physiol.  (5),  8;  Bielfeld,  Hofmeister's  Beitrage, 
2;    see  also  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39. 

1  See  Centralbl.  d.  Physiol.,  lfi,  393. 

s  See  Lapicque,  Compt.  rend.,  124,  and  Schurig,  Arch.  f.  exp.  Path.  u.  Pharm  ,  41. 

4  Arch,  do  Physiol.  (5),  10. 

'St.  Zaleski,  1.  c. ;  Kruger  and  collaborators,  Zeitschr.  f.  Biologic  27:  Lapicque, 
Maly's  Jahresber.,  20. 


244  THE  LIVER. 

Kruger  *  has  determined  the  quantity  of  calcium  in  the  liver  of  adult 
cattle  and  in  calves,  and  finds  respectively  0.71  p.  m.  and  1.23  p.  m.  of  the 
dried  substance.  In  the  foetus  of  the  cow  it  is  lower  than  in  calves.  During 
pregnancy  the  iron  and  calcium  in  the  foetus  are  antagonistic;  that  is,  an 
increase  in  the  quantity  of  calcium  in  the  liver  causes  a  diminution  in  the 
iron,  and  an  increase  in  the  iron  causes  a  decrease  in  the  calcium.  Copper 
seems  to  be  a  physiological  constituent,  and  occurs  to  a  considerable  extent' 
in  cephalopods  (Henze2).  Foreign  metals,  such  as  lead,  zinc,  and  others 
(also  iron),  are  easily  taken  up  and  retained  for  a  long  time  by  the  liver  and. 
seem  to  be  combined  with  the  nuclein  substances  (Slowtzoff,  v.  Zeynek3). 

v.  Bibra4  found  in  the  liver  of  a  young  man  who  had  suddenly  died 
762  p.  m.  water  and  238  p.  m.  solids,  consisting  of  25  p.  m.  fat,  152  p.  m. 
proteid,  gelatine-forming  and  insoluble  substances,  and  61  p.  m.  extractive 
substances. 

Glycogen  and  its  Formation. 

Glycogen  was  discovered  by  Bernard.  It  is  a  carbohydrate  closely 
related  to  the  starches  or  dextrins,  with  the  general  formula  C6H10O5,  per- 
haps 6(C6H10O5)+H2O  (KiiLZ  and  Borntrager).  The  largest  quantities 
are  found  in  the  liver,  and  smaller  quantities  in  the  muscles  (Bernard, 
Nasse).  It  is  found  in  very  small  quantities  in  nearly  all  tissues  of  the 
animal  body.  Its  occurrence  in  lymphoid  cells,  blood,  and  pus  has  been 
mentioned  in  a  previous  chapter,  and  it  seems  to  be  a  regular  constituent 
of  all  cells  capable  of  development.  Schondorff,5  who  has  determined 
the  maximum  amount  of  glycogen  in  the  dog  after  excessive  meat  and 
carbohydrate  diet,  found  7.59-37.87  grams  glycogen  per  kilo  of  the  animal. 
In  the  liver  he  found  186.9  p.  m.  glycogen  as  a  maximum.  For  100  grams 
liver-glycogen  he  found  76.17  to  398  grams  of  other  bodies.  The  muscles 
contained  7.2-37.2  p.  m.  glycogen.  Besides  the  glycogen  in  the  liver  and 
the  muscles  he  also  found  appreciable  amounts  in  the  other  organs.  Gly- 
cogen was  first  shown  to  exist  in  embryonic  tissues  by  Bernard  and  Kuhne, 
and  it  seems  on  the  whole  to  be  a  constituent  of  such  tissues  in  which  a 
rapid  cell-formation  and  cell-development  is  taking  place.  It  is  also  pres- 
ent in  rapidly  forming  pathological  swellings  (Hoppe-Seyler).  Certain 
animals,  as   certain  mussels  (Bizio),  taenia  and  ascarides    (Weinland  6),. 

1  Zeitschr.  f.  Biolo^ie,  31. 

2  Zeitschr.  f.  physiol.  Chem.,  33. 

3  Slowtzoff,  Hofmeister's  Beitrage,  1;   v.  Zeynek,  see  Ccntralbl.  f.  Physiol.,  15. 

4  See  v.  Gorup-Besanez,  Lehrbuch.  d.  physiol.  Chem.,  4.  Aufl.,  711. 
'Pfliiger's  Arch.,99. 

6  Zeitschr.  f.  Biologie,  41.  The  extensive  literature  on  Glycogen  may  be  found  in 
E.  Pfluger,  Glycogen;  in  Pfliiger's  Arch.,  96;  and  in  Cremer,  Physiol,  dcs  Glycogens  in 
Ergebnisse  der  Physiologie,  1,  Abt.  I.  In  the  following  pages  we  shall  refer  to  these 
works. 


GLYCOGEN.  245 

are  very  rich  in  glycogen.     Glycogen  also  occurs  in  the  vegetable  kingdom, 
especially  in  many  fungi. 

The  quantity  of  glycogen  in  the  liver,  as  also  in  the  muscles,  depends 
essentially  upon  the  food.  In  starvation  it  disappears  nearly  completely 
after  a  short  time,  hut  more  rapidly  in  small  than  in  large  animals,  and 
it  disappears  earlier  from  the  liver  than  from  the  muscles.  After  par- 
taking of  food,  especially  such  that  is  rich  in  carbohydrates,  the  liver  be- 
comes rich  again  in  glycogen,  the  greatest  increment  occurring  14  to  10 
hours  after  eating  (Kulz).  The  quantity  of  liver-glycogen  may  amount  to 
120-1G0  p.  m.  after  partaking  of  large  quantities  of  carbohydrates.  <  >nli- 
narily  it  is  considerably  less,  namely,  12-30  to  40  p.  m.  According  to 
Crembb  the  quantity  of  glycogen  in  plants  (yeast-cells)  is,  as  in  animals, 
dependent  upon  the  food.  According  to  him  the  yeast-cells  contain  gly- 
cogen, which  disappears  from  the  cells  in  the  auto-fermentation  of  the  yeast, 
but  reappears  on  the  introduction  of  the  cells  into  a  sugar  solution. 

The  quantity  of  glycogen  of  the  liver  (and  also  the  muscles)  is  also 
dependent  upon  rest  and  activity,  because  during  rest,  as  in  hibernation,  it 
increases,  and  during  work  it  diminishes.  Kulz  has  shown  that  by  hard 
work  the  quantity  of  glycogen  in  the  liver  (of  dogs)  is  reduced  to  a  mini- 
mum in  a  few  hours.  The  muscle-glycogen  does  not  diminish  to  the  same 
extent  as  the  liver-glycogen.  Kulz,  Zuntz  and  Vogelius,  Frentzel, 
and  others  have  been  able  to  render  rabbits  and  frogs  glycogen-free  by 
suitable  strychnine  poisoning.  The  same  result  is  produced  by  starva- 
tion followed  by  hard  work. 

Glycogen  forms  an  amorphous,  white,  tasteless,  and  inodorous  powder. 
It  gives  an  opalescent  solution  with  water  which,  when  allowed  to  evaporate 
on  the  water-bath,  forms  a  pellicle  over  the  surface  that  disappears  again 
on  cooling.  The  solution  is  dextrogyrate,  (a)D  =  +190°. 03  (Huppert). 
The  specific  rotatory  power  is  given  somewhat  differently  by  various  inves- 
tigators. A  solution  of  glycogen,  especially  on  the  addition  of  XaCl,  is 
colored  wine-red  by  iodine.  It  may  hold  cupric  hydrate  in  solution  in 
alkaline  liquids,  but  does  not  reduce  it.  A  solution  of  glycogen  in  water  is 
not  precipitated  by  potassium-mercuric  iodide  and  hydrochloric  acid,  but  is 
precipitated  by  alcohol  (on  the  addition  of  NaCl  when  necessary)  or  ammo- 
niacal  basic  lead  acetate.  An  aqueous  solution  of  glycogen  made  alkaline 
with  caustic  potash  (15  per  cent  KOH)  is  completely  precipitated  by  an 
equal  volume  of  90  per  cent  alcohol.  Tannic  acid  also  precipitates  gly- 
cogen. It  gives  a  white  granular  precipitate  of  benzoyl  glycogen  with 
benzoyl  chloride  and  caustic  soda.  Glycogen  is  completely  precipitated 
by  saturating  its  solution  at  ordinary  temperatures  with  magnesium  or 
ammonium  sulphate.  It  is  not  precipitated  by  sodium  chloride  or  half 
saturation  with  ammonium  sulphate  (Nasse,  Neumeister,  Halliburton, 


246  THE  LIVER. 

Young1).  On  boiling  with  dilute  caustic  potash  (1-2  per  cent)  the  gly- 
cogen may  be  more  or  less  changed,  especially  if  it  has  been  previously 
exposed  to  the  action  of  acid  or  of  Brucke  's  reagent  (see  below)  (Pfluger). 
On  boiling  with  stronger  caustic  potash  (even  of  36  per  cent)  it  is  not  injured 
(Pfluger).  By  diastatic  enzymes  glycogen  is  converted  into  maltose 
or  dextrose,  depending  upon  the  nature  of  the  enzyme.  It  is  transformed 
into  dextrose  by  dilute  mineral  acids.  According  to  Tebb,2  various  dex- 
trins  appear  as  intermediary  steps  in  the  saccharification  of  glycogen, 
depending  on  whether  the  hydrolysis  is  caused  by  mineral  acids  or  enzymes. 
The  question  whether  the  glycogen  from  various  animals  and  different 
organs  is  the  same  in  this  regard  has  not  been  sufficiently  investigated. 
Nor  has  it  been  decided  whether  all  the  glycogen  in  the  liver  occurs  as 
such  or  whether  it  is  in  part  combined  with  proteid  (Pfluger-Nerking). 
According  to  Seegen3  a  nitrogenous  carbohydrate  occurs  in  the  liver  and 
this  may,  according  to  him,  be  considered  perhaps  as  an  intermediary 
step  in  the  formation  of  carbohydrate  from  the  proteid. 

The  preparation  of  pure  glycogen  (simplest  from  the  liver)  is  generally 
performed  by  the  method  suggested  by  Brucke,  of  which  the  main  points 
are  the  following:  Immediately  after  the  death  of  the  animal  the  liver  is 
thrown  into  boiling  water,  then  finely  divided  and  boiled  several  times  wTith 
fresh  water.  The  filtered  extract  is  now  sufficiently  concentrated,  allowed 
to  cool,  and  the  proteids  removed  by  alternately  adding  potassium-mercuric 
iodide  and  hydrochloric  acid.  The  glycogen  is  precipitated  from  the 
filtered  liquid  by  the  addition  of  alcohol  until  the  liquid  contains  60  vols, 
per  cent.  By  repeating  this  and  precipitating  the  glycogen  several  times 
from  its  alkaline  and  acetic-acid  solution  it  is  purified  on  the  filter  by  wash- 
ing first  with  60  per  cent  and  then  with  95  per  cent  alcohol,  then  treating 
writh  ether  and  drying  over  sulphuric  acid.  It  is  always  contaminated 
writh  mineral  substances.  To  be  able  to  extract  the  glycogen  from  the 
liver  or,  especially,  from  muscles  and  other  tissues  completely,  which  is 
essential  in  a  quantitative  estimation,  these  parts  must  first  be  warmed 
for  two  hours  with  strong  caustic  potash  (30  per  cent)  on  the  water-bath. 
As  the  glycogen  changes  in  this  purification,  according  to  Brucke,  it  is 
better,  for  quantitative  determinations  of  glycogen,  to  precipitate  it  directly 
from  the  alkaline  solution  by  alcohol  (Pfluger  4). 

The  quantitative  estimation  is  best  performed  according  to  Pfluger's 
method,  which  is  based  upon  the  following:  100  grams  of  the  finely  divided 
organ  and  100  c.  c.  of  60  per  cent  caustic-potash  solution  are  heated  on  the 
water-bath  for  two  hours.  After  evaporating  the  water  to  400  c.  c.  it  is 
filtered  through  glass  wool  and  the  glycogen  precipitated  from  100  c.  c. 
of  the  filtrate  by  100  c.  c.  of  96  per  cent  alcohol.     The  glycogen  is  washed 

1  Young,  Journ.  of  Physiol.,  22,  citing  the  other  investigators. 

2  Journ.  of  Physiol.,  22. 
'Centralbl.  f.  Physiol.,  12  and  13. 

4  See  also  the  method  suggested  by  Gautier,  Comp.  rend. ,  129. 


GLYCOGEN  FORMATION.  247 

on  the  filter  first  with  dilute  alkali  and  alcohol  and  then  with  alcohol  alone. 
It  is  then  dissolved  in  water,  exactly  neutralized,  treated  with  25  c.  c. 
hydrochloric  acid  (1.19  sp.  gr.)  and  water  added  to  500  c.  c,  when  the  amount 
of  HC1  will  be  2.2  per  cent  On  heating  for  three  hours  the  glycogen  will 
have  been  converted  into  dextrose,  whose  quantity  can  be  determined 
according  to  Allihx-Pflugkk's  method  by  reduction  of  an  alkaline  copper 
solution  and  weighing  the  cuprous  oxide.  As  a  control  the  weighed  cuprous 
oxide  is  dissolved  in  nitric  acid  and  the  copper  titrated  according  to  Vol- 
hard's  method.  In  regard  to  the  detailed  steps,  which  must  be  exactly 
observed,  compare  Pfluger's  original  work.  Other  methods  of  esti- 
mating glycogen,  such  as  those  of  Brucke-Kulz,  Pavy,  and  Austin,  are 
described  in  Pfluger  's  Archiv,  96.  See  also  the  new  method  as  suggested 
by  Salkowski.1 

Numerous  investigators  have  endeavored  to  determine  the  origin  of 
glycogen  in  the  animal  body.  It  is  positively  established  by  the  unanimous 
observations  of  many  investigators  2  that  the  varieties  of  sugars  and  their 
anhydrides,  dcxtrins  and  starches,  have  the  property  of  increasing  the 
quantity  of  glycogen  in  the  body.  The  action  of  inulin  seems  to  be 
somewhat  uncertain.3  The  statements  are  questioned  in  regard  to  the 
action  of  the  pentoses.  Cremer  found  that  various  pentoses,  such  as 
rhamnose,  xylose,  and  arabinose,  have  a  positive  influence  on  the  glycogen 
formation  in  rabbits  and  hens,  and  Salkowski  obtained  the  same  result 
on  feeding  rabbits  and  a  hen  on  1-arabinose.  Frentzel  found,  on  the 
contrary,  no  glycogen  formation  on  feeding  xylose  to  a  rabbit  which  had 
previously  been  made  glycogen-free  by  strychnine  poisoning,  and  Neuberg 
and  Wohlgemuth  4  obtained  similar  negative  results  on  feeding  rabbits 
with  d-  and  r-arabinose. 

The  hexoses,  and  the  carbohydrates  derived  therefrom,  do  not  all 
possess  the  ability  of  forming  or  accumulating  glycogen  to  the  same  extent. 
Thus  C.  Voit  5  and  his  pupils  have  shown  that  -dextrose  has  a  more 
powerful  action  than  cane-sugar,  while  milk-sugar  is  less  active  (in 
rabbits  and  hens)  than  dextrose,  lsevulose,  cane-sugar,  or  maltose.  The 
following  substances  when  introduced  into  the  body  also  increase  the 
quantity  of  glycogen  in  the  liver:  glycerine,  gelatine,  arbutin,  and  likewise, 
according  to  the  investigations  of  Kulz,  crythrite,  qucrcite,  dulcitc,  man- 
nitc,  inosite,  ethylene  and  propylene  glycol,  glucuronic  anhydride,  saccharic 
acid,   mucic  acid,  sodium  tartrate,  saccharin,  isosaccharin,  and  urea.     Am- 

1  Zeitschr.  f.  physiol.  Chem.,  36. 

2  In  reference  to  the  literature  on  this  subject  see  E.  Kiilz,  Pfluger's  Arch.,  24,  and 
Luchvig-Festschrift,  1S91 ;  also  the  cited  works  of  Pfluger  and  Cremer,  foot-note  6,  page 
244. 

s  See  Miura,  Zeitschr.  f.  Biologie,  32,  and  Xakaseko,  Amer.  Journ.  of  Physiol.,  4. 
*  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  32;   Neuberg  and  Wohlgemuth,  ibid.,  35. 
See  also  Pfluger,  1.  c,  and  Cremer,  1.  c. 
'  Zeitschr.  f.  Biologie,  28. 


248  THE  LIVER. 

monium  carbonate,  glycocoll,  and  asparagin  may  similarly,  according  to 
Rohmaxx,  cause  an  increase  in  the  amount  of  glycogen  in  the  liver. 
According  to  Nebelthau  other  ammonium  salts  and  some  of  the 
amides,  as  well  as  certain  narcotics,  hypnotics,  and  antipyretics,  produce 
an  increase  in  the  glycogen  of  the  liver.  This  action  of  the  anti- 
pyretics    (especially     antipyrin)     had     been     shown     by     Lepixe     and 

PORTERET. * 

The  fats,  according  to  Bouchard  and  Desgrez,  increase  the  glycogen 
content  of  the  muscles,  but  not  of  the  liver,  and  according  to  Couvreur  2 
the  glycogen  is  increased  at  the  expense  of  the  fat  in  the  silkworm  larva 
as  it  changes  into  a  chrysalis.  In  general  it  is  believed  that  the  fat  has 
no  influence  upon  the  glycogen  content  of  the  liver,  although  glycerine 
has  the  action  above  mentioned. 

The  views  in  regard  to  the  influence  of  the  proteids  are  somewhat  con- 
tradictory. From  several  investigations  the  conclusion  has  been  drawn 
that  the  proteids  cause  an  increase  in  the  glycogen  of  the  liver.  Amongst 
these  investigations  must  be  included  certain  feeding  experiments  with 
boiled  beef  (Nauxtx)  or  blood-fibrin  (v.  Merixg),  and  especially  the 
very  careful  experiments  made  by  E.  Kulz  on  hens  with  pure  proteids, 
such  as  casein,  seralbumin,  and  ovalbumin.  The  value  of  these  experi- 
ments is  disputed  by  Pfluger,  and  as  a  direct  proof  against  the  formation 
of  glycogen  from  proteid  he  refers  to  Schoxdorff's  investigations  when 
feeding  carbohydrate-free  proteid  (casein)  to  frogs  without  finding  the  least 
increase  in  the  total  glycogen.  Later  Blumexthal  and  Wohlgemuth 
arrived  at  similar  results.  They  found  no  glycogen  accumulation  in  frogs 
after  feeding  with  casein  or  gelatine,  but  did  find  it  after  feeding  with  oval- 
bumin, which  contains  a  carbohydrate  group.  On  the  contrary,  Bexdix 
was  able  to  show  an  increase  in  the  glycogen  in  dogs  by  feeding  casein 
and  gelatine,  as  well  as  ovalbumin,  and  in  fact  a  greater  increase  by 
casein  than  by  ovalbumin.  Stookey3  arrived  at  similar  results  when  he 
found  in  hens  a  glycogen  formation  after  feeding  casein,  while  he  obtained 
no  positive  results  after  feeding  glucoproteids.  It  seems  as  if  the  condi- 
tions in  cold-blooded  animals  were  different  from  those  in  warm-blooded 
ones.  According  to  Pfluger,  the  experiments  of  Bexdix  are  not  con- 
clusive, and  he  doubts  the  formation  of  glycogen  from  proteid.  He  claims 
it  is  only  formed  from  carbohydrates  or  from  the  carbohydrate  complex 

1  Rohmann,  Pfluger 's  Arch.,  39;  Nebelthau,  Zeitschr.  f.  Biologie,  28;  Porteret, 
Compt.  rend.,  106. 

2  Bouchard  et  Desgrez,  Compt.  rend.,  130;  Couvreur,  Compt.  rend,  de  Soc.  biol., 
47. 

'  Schondorff,  Pfluger 's  Arch.,  82  and  88;  Blumenthal  and  Wohlgemuth,  Berl.  klin. 
Wochenschr. ,  1901;  Bendix,  Zeitschr.  f.  physiol.  Chem.,  32  and  34;  Stookey,  Amer. 
Journ.  of  Physiol.,  9. 


GLYCOGEN  FORMATION.  249 

of  the  glucoproteids.     Most  investigators  are  still,  it  seems,  of  the  opinion 
that  glycogen  can  be  produced  from  carbohydrate-free  proteids. 

If  the  question  is  raised  as  to  the  action  of  the  various  bodies  on  the 
accumulation  of  glycogen  in  the  liver,  it  must  be  recalled  that  a  forma- 
tion of  glycogen  takes  place  in  this  organ,  as  well  as  a  consumption  of 
the  same.  An  accumulation  of  glycogen  may  be  caused  by  an  increased 
formation  of  glycogen,  but  also  by  a  diminished  consumption,  or  by 
both. 

It  is  not  known  how  the  various  bodies  above  mentioned  act  in  this 
regard.  Certain  of  them  probably  have  a  retarding  action  on  the  transfor- 
mation of  glycogen  in  the  liver,  while  others  perhaps  are  more  combustible 
and  in  this  way  protect  the  glycogen.  Some  probably  excite  the  liver-cells 
to  a  more  active  glycogen  formation,  while  others  yield  material  from  which 
the  glycogen  is  formed  and  are  glycogen  formers  in  the  strictest  sense  of  the 
word.  The  knowledge  of  these  last-mentioned  bodies  is  of  the  greatest, 
importance  in  the  question  as  to  the  origin  of  glycogen  in  the  animal  body, 
and  the  chief  interest  attaches  itself  to  the  question,  to  what  extent  are 
the  two  chief  groups  of  food,  the  proteids  and  carbohydrates,  glycogen 
formers? 

The  great  importance  of  the  carbohydrates  in  the  formation  of  glycogen 
has  given  rise  to  the  opinion  that  the  glycogen  in  the  liver  is  produced  from 
sugar  by  a  synthesis  in  which  water  separates  with  the  formation  of  an 
anhydride  (Luchsinger  and  others).  This  theory  (anhydride  theory) 
has  found  opponents  because  it  neither  explains  the  formation  of  glycogen 
from  such  bodies  as  proteids,  carbohydrates,  gelatine,  and  others,  nor 
the  circumstance  that  the  glycogen  is  always  the  same  independent  of 
the  properties  of  the  carbohydrate  introduced,  whether  it  is  dextrogyrate 
or  lsevogyrate.  It  is  therefo.e  the  opinion  of  many  investigators  that 
all  glycogen  is  formed  from  proteid,  and  that  this  splits  into  two  parts, 
one  containing  nitrogen  and  the  other  being  free  from  nitrogen:  the  latter 
is  the  glycogen.  According  to  these  views,  the  carbohydrates  act  only  in 
that  they  spare  the  proteid  and  the  glycogen  produced  therefrom  (sparing- 
theory  of  Weiss,  Wolffberg,  and  others1). 

In  opposition  to  this  theory  C.  and  E.  Voit  and  their  pupils  have  shown 
that  the  carbohydrates  are  "true"  glycogen  formers.  After  partaking 
of  large  quantities  of  carbohydrates  the  amount  of  glycogen  stored  up 
in  the  body  is  sometimes  so  great  that  it  cannot  be  covered  by  the  proteids 
decomposed  during  the  same  time,  and  in  these  cases  a  glycogen  formation 
from  the  carbohydrates  must  be  admitted.  According  to  Cremeb  only  the 
fermentable  sugar  of  the  six  carbon  series  or  their  di-  and  polysaccharides 
are  true  glycogen  formers.     For  the  present  one  only  designates  dextrose, 

1  In  regard  to  these  two  theories,  see  especially  Wolffberg,  Zeitschr.  f.  Diologie,  16. 


250  THE  LIVER. 

lsevulose,  galactose  (Weinland  l)  and  perhaps  also  d-mannose  (Cremer) 
as  true  glycogen  formers.  Other  monosaccharides  may  indeed,  according 
to  Cremer,  influence  the  formation  of  glycogen,  but  they  are  not  converted 
into  glycogen  and  hence  are  only  called  pseudoglycogen  formers. 

The  poly-  and  disaccharides  may,  after  a  cleavage  into  the  correspond- 
ing fermentable  monosaccharides,  serve  as  glycogen  formers.  This  is  true 
for  at  least  cane-sugar  and  milk-sugar,  which  must  first  be  inverted  in  the 
intestine.  These  two  varieties  of  sugar,  therefore,  cannot,  like  dextrose  and 
lsevulose,  serve  as  glycogen  formers  after  subcutaneous  injection,  but  re- 
appear nearly  entirely  in  the  urine  (Dastre,  Fr.  Voit).  Maltose,  which  is 
inverted  by  an  enzyme  present  in  the  blood,  passes  only  to  a  slight  extent 
into  the  urine  (Dastre  and  Bourquelot  and  others),  and  it  can,  like  the 
monosaccharides,  even  after  subcutaneous  injection,  be  used  in  the  formation 
of  glycogen  (Fr.  Voit2). 

After  Pavy  3  showed  the  glucoproteid  nature  of  ovalbumin  and,  as  shown 
later,  that  glucosamine  could  be  split  off  from  ovalbumin  as  well  as  certain 
other  protein  substances  (see  Chapter  II),  the  question  arose  whether  the 
amino-sugar  could  serve  in  the  formation  of  glycogen.  The  investigations 
carried  out  in  this  direction  by  Fabian,  Frankel  and  Offer,  and  Cathcart  4 
have  shown  that  the  glucosamine  introduced  into  the  organism  is  in  part 
eliminated  unchanged  in  the  urine  and  has  no  glycogen-forming  action. 
No  definite  conclusions  can  be  drawn  from  this  on  the  behavior  of  the 
carbohydrate  groups  which  exist  not  as  free  groups  but  combined  with 
the  proteid  molecules. 

Whether  or  not,  or  to  what  extent,  the  glucoproteids  take  part  in  the 
sugar  of  glycogen  formation  in  the  animal  body  is  difficult  to  answer  for 
the  present,  as  but  little  is  known  of  the  extent  of  these  substances  in  the 
body  and  our  knowledge  of  the  amount  of  carbohydrate  which  can  be  split 
off  from  the  various  protein  substances  is  also  very  meagre.  The  most 
widely  accepted  view  seems  to  be  that  the  quantity  of  sugar  eliminated 
under  certain  conditions — in  several  cases  of  diabetes  of  different  kinds — was 
too  great  to  be  covered  by  the  glycogen  content  of  the  body  and  the  glu- 
coproteids, and  in  these  cases  a  sugar  formation  from  proteid  is  admitted. 

The  greatest  quantity  of  sugar  which  could  be  formed  theoretically 
from  proteid  Is  8  grams  of  sugar  for  every  gram  of  proteid  nitrogen  if  it 

1  E.  Voit,  Zeitschr.  f.  Biologie,  25,  543,  and  C.  Voit,  ibid.,  28.  See  also  Kausch 
and  Socin,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Weinland,  Zeitschr.  f.  Biologie,  40  and 
88;  Cremer,  ibid.,  42,  and  Ergebnisse  dcr  Physiol.,  1. 

2  Dastre,  Arch,  de  Physiol.  (5),  3,  1891;  Dastre  and  Bourquelot,  Compt.  rend.,  98; 
Fritz  Voit,  Verhandl.  d.  Gesellsch.  f.  Morph.  u.  Physiol,  in  Miinchen,  1896,  and  Deutsch. 
Arch.  f.  klin.  Med.,  58. 

3  The  Physiology  of  the  Carbohydrates,  London,  1894. 

*  Fabian,  Zeitschr.  f.  physiol.  Chem.,  27;  Frankel  and  Offer,  Centralbl.  f.  Physiol., 
13;  Cathcart,  Zeitschr.  f.  physiol.  Chem.,  39. 


GLYCOGEN  FORMATION.  251 

is  admitted  that  all  the  carbon  of  the  proteid,  with  the  excepl  ion  of  that  nec- 
essary to  form  ammonium  carbonate,  is  used  in  the  formation  of  sugar.  The 
relationship  between  dextrose  and  nitrogen  in  the  urine  has  been  repeatedly 
determined  in  various  forms  of  diabetes.  Minkowski  and  a  few  other 
investigators1  have,  after  meat  feeding  in  cases  of  artificial  pancreas  diabetes, 
found  the  ratio  2.8-3:1  and  in  phlorhizin  diabetes  D  :N  =  3.8-4.2:1.2  In 
human  diabetes  still  higher  results  for  the  sugar  elimination  have  been 
found,  and  indeed  in  a  few  cases  with  food  as  poor  in  carbohydrates  as 
possible  the  ratio  higher  than  8:1  was  observed.  There  are  indeed  cases 
where  the  large  quantities  of  sugar  eliminated  cannot  be  accounted  for 
by  the  calculated  carbohydrate  and  proteid  transformation,  but  it  is  found 
necessary  to  admit  of  a  sugar  formation  from  fats — a  view  that  is  not  based 
upon  sufficiently  conclusive  observations. 

It  does  not  seem  justifiable  to  draw  positive  conclusions  from  the  size 
of  the  sugar  elimination  and  from  the  ratio  D :  N,  irrespective  of  those 
cases  where  evident  faults  are  present.  On  the  other  hand,  we  do  not 
know  the  glycogen  condition  of  the  individual  experimented  upon  nor 
the  amount  of  sugar  split  off  from  the  glucoproteids,  and  also  it  is  not  possi- 
ble to  estimate  the  amount  of  sugar  formed  from  the  quantity  of  sugar 
eliminated  by  the  urine,  as  an  unknown  part  of  the  sugar  is  undoubtedly 
burnt  in  the  body.  The  ordinary  view  is,  as  above  stated,  that  a  sugar 
formation  and — what  amounts  to  the  same  thing — a  glycogen  formation 
from  proteid  has  been  positively  shown. 

One  can  only  speculate  on  the  manner  in  which  the  sugar  is  formed 
from  the  proteid.  It  is  still  generally  admitted  that  a  deep  cleavage  first 
takes  place.  Fr.  Muller  has  proposed  the  view  that  the  sugar  formation 
comes  possibly  from  the  leucin,  a  view,  although  it  has  been  the  subject 
of  several  investigations  (R.  Cohn,  Luthje,  Bendix,  Schondorff,  Blu- 
menthal,  and  Wohlgemuth  3),  has  not  been  proven  nor  positively  dis- 
proved. 

Like  the  carbohydrates  in  general,  glycogen  has  without  any  doubt  a 
great  importance  in  the  formation  of  heat  and  development  of  energy  in 
the  animal  body.  The  possibility  of  the  formation  of  fat  from  glycogen 
cannot  be  denied.  *  Glycogen  is  generally  considered  as  accumulated  reserve 
food  in  the  liver  and  formed  in  the  liver-cells.  Where  does  the  glycogen 
existing  in  the  other  organs,  such  as  the  muscles,  originate?     Is  the  gly- 

1  Arch.  f.  exp.  Path.  u.  Pharm.,  31.  As  the  scope  of  this  work  does  not  allow  of 
the  reference  to  the  extensive  literature  on  the  elimination  of  sugar  in  the  various 
forms  of  diabetes,  the  reader  is  referred  to  larger  handbooks  and  monographs  on  diabetes. 

'Stiles  and  Lusk,  Amer.  Journ.  of  Physiol.,  10. 

'Cohn,  Zeitschr.  f.  physiol.  Chem.,  28;  Bendix,  ibid.,  32;  Luthje,  Zeitschr.  f.  klin. 
Med.,  39;  Schondorff,  Pfluger's  Arch.,  82;  Blumenthal  and  Wohlgemuth,  Berl.  klin. 
Wochenschr.,  1901;  Simon,  Zeitschr.  f.  physiol.  Chem.,  35. 

*  See  especially  Nocl-Paton,  Journ.  of  Physiol.,  19. 


252  THE  LIVER. 

cogen  of  the  muscles  formed  on  the  spot  or  is  it  transmitted  to  the  muscles 
by  the  blood?  These  questions  cannot  yet  be  answered  with  positiveness, 
and  the  investigations  on  this  subject  by  different  experimenters  have 
given  contradictory  results.  The  experiments  of  Kulz,1  in  which  he 
studied  the  glycogen  formation  by  passing  blood  containing  cane-sugar 
through  the  muscle,  has  led  to  no  conclusive  results.  Still  the  formation 
of  glycogen  from  sugar  in  the  muscles  is  probable.  There  is  no  doubt  that 
glycogen  is  formed  in  the  muscles  during  embryonic  life. 

If  it  is  true  that  the  blood  and  lymph  contain  a  diastatic  enzyme  which 
transforms  glycogen  into  sugar,  and  also  that  the  glycogen  regularly  occurs 
in  the  form-elements  and  is  not  dissolved  in  the  fluids,  it  seems  probable 
that  the  glycogen  is  not  transmitted  by  the  blood  to  the  organs  in  solu- 
tion, but  perhaps  more  likely,  if  the  leucocytes  do  not  act  as  carriers,  it 
is  formed  on  the  spot  from  the  sugar.2  The  glycogen  formation  seems  to 
be  a  general  function  of  the  cells.  In  adults  the  liver,  which  is  very  rich 
in  cells,  has  the  property,  on  account  of  its  anatomical  position,  of  trans- 
forming large  quantities  of  sugar  into  glycogen. 

The  question  now  arises  whether  there  is  any  foundation  for  the  state- 
ment that  the  liver  glycogen  is  transformed  into  sugar. 

As  first  shown  by  Bernard  and  repeated  by  many  investigators,  the 
glycogen  in  a  dead  liver  is  gradually  changed  into  sugar,  and  this  sugar 
formation  is  caused,  as  Bernard  supposed  and  Arthus  and  Huber,  Pavy, 
and  recently  also  Pick  and  Bial,3  proved,  by  a  diastatic  enzyme.  This  post- 
mortem sugar  formation  led  Bernard  to  the  assumption  of  the  formation 
of  sugar  from  glycogen  in  the  liver  during  life.  Bernard  suggested  the 
following  arguments  for  this  theory:  The  liver  always  contains  some  sugar 
under  physiological  conditions,  and  the  blood  from  the  hepatic  vein  is 
always  somewhat  richer  in  sugar  than  the  blood  from  the  portal  vein. 
The  correctness  of  either  or  both  of  these  statements  has  been  disputed 
hy  many  investigators.  Pavy,  Ritter,  Schifp,  Eulenberg,  Lussana, 
Abeles,  and  others  deny  the  occurrence  of  sugar  in  the  liver  during  life, 
and  the  greater  amount  of  dextrose  in  the  blood  from  the  hepatic  vein  is 
likewise  disputed  by  them  and  certain  other  investigators.4 

It  can  be  said  that  at  present  there  are  cwo  chiei  views  on  the  destruc- 
tion of  the  glycogen  in  the  living  organism:  Pavy's  view,  that  the  glycogen 

1  See  Minkowski  and  Laves,  Arch.  f.  exp.  Path.  u.  Pharm.,  23;  Kiilz,  Zeitschr.  f. 
Biologie,  2" 

2  See  Dastre,  Compt.  rend,  de  soc.  biol.,  -47,  280,  and  Kaufmann,  ibid.,  316. 

3  Arthus  and  Huber,  Arch,  de  Physiol.  (5),  4,  659;  Pavy,  Journal  of  Physiol.,  22; 
Pick,  Hofmeister's  Beitr.,  3;  Bial,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 

4  In  regard  to  the  literature  on  sugar  formation  in  the  liver  see  Bernard,  Lecons  sur 
ie  diabete,  Paris,  1877;  Seegen,  Die  Zuckerbildung  im  Tierkorper,  Berlin,  1890;  M. 
Bial,  Pfluger's  Arch.,  55,  434. 


FORMATION  OF  SUGAR  IN  THE  LIVER.  253 

is  directly  used  without  being  previously  transformed  into  sugar,  and  Ber- 
nard 's  view,  which  is  accepted  by  most  investigators,  that  the  glycogen  is 
first  transformed  into  sugar  by  the  aid  of  diastatic  enzymes.  According 
to  certain  experimenters  (Dastre,  Noel-Paton,  E.  Cavazzani  '),  who 
also  admit  a  destruction  of  the  glycogen  with  the  formation  of  sugar,  the 
change  is  not  brought  about  by  an  enzyme  but  by  a  special  protoplasmic 
activity. 

The  doctrine  as  to  the  physiological  formation  of  sugar  in  the  liver  has 
obtained  an  energetic  advocate  in  Seegen.  He  maintains,  after  numerous 
experiments,  that  the  liver  regularly  contains  considerable  amounts  of 
sugar.  He  has  observed  an  increase  of  3  per  cent  in  the  quantity  of  dex- 
trose in  the  liver  of  a  dog  kept  alive  by  passing  arterial  blood  through  the 
organ,  and  lastly  he  has  also  found  in  a  very  great  number  of  experiments 
on  dogs  that  the  blood  from  the  hepatic  vein  always  contains  more — even 
double  as  much — sugar  than  the  blood  from  the  portal  vein.  Mosse  and 
Zuntz  2  have  recently  made  objections  as  to  the  correctness  of  this  last 
statement,  and  it  follows  from  the  various  researches  on  this  question 
that  when  disturbing  influences  are  prevented  the  blood  from  the  hepatic 
vein  is  only  very  little  richer  in  sugar  than  the  blood  from  the  portal  vein. 

Although  Seegen  energetically  espouses  the  doctrine  of  Bernard  as  to 
the  vital  sugar  formation  in  the  liver,  still  he  deviates  essentially  from 
Bernard  in  that  he  claims  the  sugar  is  not  derived  from  the  glycogen. 
According  to  Seegen  the  sugar  is  formed  from  proteid  and  fat.  His 
older  idea,  that  this  proteid  was  peptone,  he  has  discarded.  Of  importance 
for  the  study  of  the  sugar  formation  in  the  liver  is,  on  the  contrary,  the 
fact  that  Seegen  has  found  a  substance  in  the  liver,  besides  glycogen, 
which  yields  dextrose  on  heating  with  dilute  acids.  He,  in  connection  with 
Niemann,  has  isolated  this  substance  in  the  form  of  a  nitrogenous  car- 
bohydrate. O.  Simon  3  has  also  recently  isolated  a  proteose-like  sub- 
stance from  the  liver,  which  reduces  directly  and  yields  a  fermentable 
sugar  on  boiling  with  acids,  and  this  sugar  gives  an  osazone  melting  at  190°. 

The  formation  of  carbohydrate  or  dextrose  from  fat,  a  process  which 
undoubtedly  occurs  in  the  vegetable  kingdom,  is  also  admitted  for  the 
animal  body  by  French  experimenters,  especially  Chauveau  and  Kauf- 
mvnw4  At  present  there  is  no  positively  conclusive  proof  for  such  a 
view.  As  proof  of  the  formation  of  sugar  from  fat  several  cases  of  dia- 
betes have  been  observed  recently  (Rumpf,  Rosenqvist,  Mohr,  v.  Noor- 

1  In  regard  to  the  literature  see  Pick,  Hofmeister's  Beitriige,  3. 

2  Seegen,  Die  Zuckerbildung,  etc.,  and  Centralbl.  f.  Physiol.,  10,  497  and  822} 
Zuntz,  ibid.,  561;  Mosse,  Pfluger's  Arch.,  63;  Bing,  Skand.  Arch.  f.  Physiol.,  9. 

3  Seegen,  Arch.  f.  (Anat.  u.)  Physiol.,  1903;  Seegen  and  Niemann,  Wien.  Sitzber., 
112  (1903^;   Simon,  Arch.  f.  (Anat.  u.)  Physiol.,  49. 

4  Kaufmann,  Arch,  de  Physiol.  (5),  8,  which  also  cites  Chauveau. 


254  THE  LIVER. 

den,  and  others  x)  in  man,  and  by  Hartogh  and  Schumm  also  in  animals, 
in  which  the  sugar  elimination  as  compared  to  the  nitrogen  elimination  for 
the  same  time  was  so  exceedingly  high  that  they  were  obliged  to  admit 
of  a  sugar  formation  from  fat;  still  these  observations  do  not  seem  to 
be  sufficiently  conclusive.  The  investigations  of  J.  Weiss  seem  to  show 
a  formation  of  sugar  from  fat  in  the  liver,  while,  on  the  contrary,  the  obser- 
vations of  Montuori  contradict  such  a  process.2  This  question  is  therefore 
disputed. 

The  circumstance  that  the  blood-sugar  rapidly  sinks  to  £-$•  of  its  orig- 
inal quantity,  or  even  disappears  when  the  liver  is  cut  out  of  the  circulation, 
speaks  for  a  vital  formation  of  sugar  in  the  liver  (Seegen,  Bock,  and 
Hoffmann  Kaufmann  Tangl  and  Harley  Pavy).  In  geese  whose 
livers  were  removed  from  the  circulation  Minkowski  found  no  sugar  in 
the  blood  after  a  few  hours.  On  removing  the  liver  from  the  circulation 
by  tying  all  the  vessels  to  and  from  the  organ,  the  quantity  of  sugar  in  the 
blood  on  drawing  is  not  increased  (Schenck3).  We  will  also  learn  shortly 
of  certain  poisons  and  operative  changes  which  may  cause  an  abundant 
elimination  of  sugar,  but  only  when  the  liver  contains  glycogen.  If  we  re- 
call the  fact  shown  by  Rohmann  and  Bial  4  that  the  lymph  as  well  as  the 
blood  contains  a  diastatic  enzyme,  then  several  reasons  speak  for  the  view 
of  Bernard,  that  the  post-mortem  formation  of  sugar  from  the  glycogen 
in  the  liver  is  a  continuation  of  the  vital  process. 

The  relationship  of  the  sugar  eliminated  in  the  urine  under  certain 
conditions,  such  as  in  diabetes  mellitus,  certain  intoxications,  lesions  of 
the  nervous  system,  etc.,  to  the  glycogen  of  the  liver  is  also  an  important 
question. 

It  does  not  enter  into  the  plan  and  scope  of  this  book  to  discuss  in 
detail  the  various  views  in  regard  to  glycosuria  and  diabetes.  The  appear- 
ance of  dextrose  in  the  urine  is  a  symptom  which  may  have  essentially 
different  causes,  depending  upon  different  circumstances.  Only  a  few  of 
the  most  important  points  will  be  mentioned. 

The  blood  contains  always  about  an  average  of  1.5  p.  m.,  while  the 
urine  at  most  only  traces  of  dextrose.  When  the  quantity  of  sugar  in  the 
blood  rises  to  3  p.  m.  or  above,  then  sugar  passes  into  the  urine.  The 
kidneys  have  the  property  to  a  certain  extent  of  preventing  the  passage  of 
blood-sugar  into  the  urine;  and  it  follows  from  this  that  an  elimination  of 

1  Rumpf,  Berl.  klin.  Wochenschr. ,  1899;  Rosenqvist,  ibid.;  Mohr,  ibid.,  1901; 
v.  Noorden,  "Die  Zuckerkrankheit,"  3.  Aufl.,  Berlin,  1901;  Hartogh  and  Schumm, 
Arch.  f.  exp.  Path.  u.  Pharm.,  47,  and  Lusk,  Zeitschr.  f.  Biologie,  42. 

2  Weiss,  Zeitschr.  f.  physiol.  Chem.,  24;  Montuori,  Maly's  Jahresber.,  26. 

3  Seegen,  Bock  and  Hoffmann,  see  Seegen,  1.  c. ;  Kaufmann,  Arch,  de  Physiol.  (5), 
8;  Tangl  and  Harley,  Pfliiger's  Arch.,  61;  Pavy,  Journ.  of  Physiol.,  29;  Minkowski, 
Arch.  f.  exp.  Path.  u.  Pharm.,  21;  Schenck,  Pfliiger's  Arch.,  57. 

*  Rohmann  and  Bial,  see  foot-note  4,  page  155. 


GLYCOSURIA.  256 

sugar  in  the  urine  may  be  caused  partly  by  a  reduction  or  suppression 
of  this  above-mentioned  activity  and  partly  also  by  an  abnormal  increase 
of  the  quantity  of  sugar  in  the  blood. 

The  first  seems,  according  to  v.  Mkring  and  Minkowski,  to  be  the 
case  in  phlorhizin  diabetes,  v.  Mering  has  found  that  a  strong  glycosuria 
appears  in  man  and  animals  on  the  administration  of  the  ghicoside  phlor- 
hizin. The  sugar  eliminated  is  not  derived  from  the  glucoside  alone.  It 
is  formed  in  the  animal  body,  and  in  fact,  at  least  on  prolonged  starvation, 
from  the  protein  substances  of  the  body.  The  quantity  of  sugar  in  the 
blood  is  not  increased,  but  rather  diminished,  in  phlorhizin  diabetes  (Min- 
kowski), which  tends  to  show  that  an  abnormal  elimination  of  sugar  takes 
place  through  the  kidneys.  This  statement  is  disputed  by  certain  investi- 
gators, Levene  and  Pavy,  and  the  work  of  the  latter  seems  to  show  that 
a  sugar  formation  takes  place  in  the  kidneys.1 

With  the  exception  of  phlorhizin  diabetes,  which  is  dependent,  accord- 
ing to  the  ordinary  views,  upon  a  change  in  the  kidneys,  all  other  forms  of 
glycosuria  or  diabetes,  as  far  as  known  at  present,  depend  on  a  hyperglu- 
ccemia . 

A  hyperglucaemia  may  be  caused  in  various  ways.  It  may  be  caused, 
for  example,  by  the  introduction  of  more  sugar  than  the  body  can  destroy. 

The  property  of  the  animal  body  to  assimilate  the  different  varieties 
of  sugar  has  naturally  a  limit.  If  too  much  sugar  is  introduced  into  the 
intestinal  tract  at  one  time,  so  that  the  so-called  assimilation  limit  (see 
Chapter  IX,  on  absorption)  is  overreached,  then  the  excess  of  absorbed 
sugar  passes  into  the  urine.  This  form  of  glycosuria  is  called  alimentary 
glycosuria,2  and  it  is  caused  by  the  passage  of  more  sugar  into  the  blood 
than  the  liver  and  other  organs  can  destroy. 

As  the  liver  cannot  transform  all  the  sugar  into  glycogen  which  comes 
to  it  in  alimentary  glycosuria,  it  is  possible  that  a  glycosuria  may  be  pro- 
duced also  under  pathological  conditions,  even  by  a  medium  amount  of 
carbohydrate  (100  grams  dextrose),  which  a  healthy  person  could  overcome. 
This  is  the  case  among  others  in  various  affections  of  the  cerebral  system 
and  in  certain  chronic  poisoning.  Seegen  includes  the  lighter  forms  of 
diabetes  in  this  class  of  glycosuria. 

1  In  regard  to  the  literature  on  phlorhizin  diabetes  see:  v.  Mering,  Zeitschr.  f.  klin. 
Med.,  14  and  10;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Moritz  and  Prausnitz, 
Zeitschr.  f.  Biologic,  27  and  29;  Kiilz  and  Wright,  ibid.,  27,  181;  Cremer  and  Hitter, 
ibid.,  28  and  29;  Contjean,  Compt.  rend,  de  soc.  biol.,  4S;  Lusk,  Zeitschr.  f.  Biologie, 
36;  Levene,  Journal  of  Physiol.,  17;  Pavy,  ibid.,  20  and  29;  Artcaga,  Amer.  Jo  irn. 
of  Physiol.,  6;  O.  Loewi,  1.  c. ;  Cremer,  Ergebnisse  der  Physiologie,  1,  Abt.  1,  and  the 
monographs  upon  diabetes;  Stiles  and  Lusk,  Amer.  Journ.  of  Physiol.,  10. 

3  In  regard  to  alimentary  glycosuria  see  Moritz,  Arch.  f.  klin.  Med.,  40,  which  also 
contains  the  older  literature;  B.  Rosenberg,  "Ueber  das  Vorkommen  der  alimentaren 
Glykosoria, "  etc.  (Inaug.-Dissert.  Berlin,  1897);  van  Oordt,  Munch,  med.  Wochen- 
schr.,  1898;  v.  Xoorden,  Die  Zuckerkrankheit,  3.  Aufl..  1901. 


256  THE  LIVER. 

We  differentiate  between  light  and  severe  forms  of  diabetes.  In  the 
first  the  urine  contains  sugar  only  when  carbohydrates  are  taken  as  food, 
while  in  the  other  case  the  urine  contains  sugar  even  with  food  entirely 
free  from  carbohydrates.  According  to  the  views  of  several  investigators, 
in  light  forms  of  diabetes  the  liver  is  incapable  of  transforming  all  the 
carbohydrates  introduced  into  glycogen,  or  to  utilize  this  in  a  normal  way, 
and  the  activity  of  the  liver-cells  is  also  reduced  or  changed  in  these  cases. 

A  hyperglucsemia  which  passes  into  a  glycosuria  may  also  be  brought 
about  by  an  excessive  formation  of  sugar  from  the  glycogen  and  other 
bodies  within  the  animal  body. 

The  so-called  piqtire,  and  also  probably  those  glycosurias  which  occur 
after  other  lesions  of  the  nervous  system,  belong  to  the  above  group  of 
glycosurias.  The  glycosuria  produced  on  poisoning  with  carbon  monox- 
ide, adrenalin,  curare,  strychnine,  morphine,  etc.,  also  belongs  to  this  group. 
That  the  glycosuria  produced  in  certain  cases,  as  after  piqiire,  is  due 
to  an  increased  transformation  of  the  glycogen  follows  from  the  fact  that 
no  glycosuria  appears,  under  the  above-mentioned  circumstances,  when 
the  liver  has  been  previously  made  free  from  glycogen  by  starvation  or 
other  means.  In  other  cases,  as  in  carbon-monoxide  poisoning,  the  sugar 
is  probably  derived  from  the  proteids,  because  glycosuria  only  occurs  in 
those  cases  where  the  poisoned  animal  has  a  sufficient  quantity  of  proteid 
at  its  disposal  (Straub  and  Rosenstein  *).  Proteid  starvation  with  a 
simultaneously  abundant  supply  of  carbohydrates  causes  this  glycosuria  to 
disappear. 

A  hyperglucsemia  with  glycosuria  may  also  be  caused  by  a  decreased 
ability  of  the  animal  body  to  consume  or  destroy  the  sugar.  In  this  case 
the  sugar  must  accumulate  in  the  blood,  and  the  formation  of  severe  cases 
of  diabetes  mellitus  is  now  generally  explained  by  this  process. 

The  inability  of  diabetics  to  destroy  or  consume  the  sugar  does  not  seem 
to  be  connected  with  any  decrease  in  the  oxidative  energy  of  the  cells. 
Apart  from  the  fact  that  the  oxidative  processes  are  not  diminished  generally 
in  diabetics  (Schultzen,  Nencki  and  Sieber  2),  it  must  be  remarked  that 
the  two  varieties  of  sugar,  dextrose  and  laevulose,  which  are  oxidized  with 
the  same  readiness,  act  differently  in  diabetics.  According  to  Kulz  and 
other  investigators  laevulose  is,  contrary  to  dextrose,  utilized  to  a  great 
extent  in  the  organism,  and  may,  according  to  Minkowski,3  even  cause  a 

1  See  Bock,  Pfliiger's  Arch.,  5;  Bock  and  Hoffmann,  Expt.  Studien  iiber  Diabetes 
(Berlin,  1874) ;  CI.  Bernard,  Lecons  sur  le  diabete  (Paris) ;  T.  Araki,  Zeitschr.  f.  physiol. 
Chem.,  15,  351;  Straub,  Arch.  f.  exp.  Path.  u.  Pharm.,  38;  Rosenstein,  ibid.,  40; 
Pfliiger,  Pfliiger's  Arch.,  96. 

2  Schultzen,  Berl.  klin.  Wochenschr. ,  1872;  Nencki  and  Sieber,  Journ.  f.  prakt. 
Chem.  (N.  F.),  26,  35. 

3  Kulz,  Beitrage  zur  Path.  u.  Therap.  des  Diabetes  mellitus  (Marburg,  1874),  1; 


DIABETES.  257 

deposit  of  glycogen  in  the  liver  in  animals  with  pancreas  diabetes  (see  be- 
low). The  combustion  of  proteid  and  fat  takes  place  as  in  healthy  subjects, 
and  the  fat  is  completely  burnt  into  carbon  dioxide  and  water.  In  this 
diabetes  the  ability  of  the  cells  to  utilize  especially  the  dextrose  suffers 
diminution,  and  the  explanation  of  this  has  been  sought  in  the  fact  that  the 
dextrose  is  not  previously  split  before  combustion. 

CO 
The  variation  in  the  respiratory  quotient,  i.e.,  the  relation  — -,  seems 

to  show  an  insufficiency  of  the  dextrose  combustion  in  the  tissues  in  diabetes. 
As  will  be  thoroughly  explained  in  a  following  chapter,  this  quotient  is 
greater  the  more  carbohydrates  are  burnt  in  the  body,  and  it  is  correspond- 
ingly smaller  when  proteid  and  fat  are  chiefly  burnt.  The  investigations 
of  Leo,  Hanriot,  Wkintraud  and  Laves,  Struve,1  and  others  have 
shown  that  in  severe  cases  of  diabetes  in  the  starving  condition  the  low 
quotient  is  not  raised  after  partaking  dextrose,  as  in  healthy  individuals,  but 
that  it  is  raised  after  feeding  laevulose,  which  is  also  of  value  to  diabetics 
(Weixtraud  and  Laves,  Struve).  The  poverty  of  the  organs  and  tis- 
sues of  diabetics  in  glycogen  shows  that  not  only  is  the  combustion  of  the 
dextrose  diminished,  but  also  the  transformation  of  the  same  into  glycogen, 
and  its  valuation  as  a  whole  is  decreased. 

There  are  also  certain  investigators  who  consider  that  diabetes  is  due  to 
an  increased  production  of  sugar  in  the  liver — a  view  which  has  received 
some  support  in  the  artificially  produced  pancreatic  diabetes  (Chauveau, 
Kaufmanx,  Cavazzaxi) . 

The  investigations  of  Minkowski,  v.  Mering,  Domenicis,  and  later 
investigators  2  have  shown  that  a  true  diabetes  of  a  severe  kind  is  caused 
by  the  total  extirpation  of  the  pancreas  of  many  animals,  especially  dogs. 
As  in  man  in  severe  forms  of  diabetes,  so  also  in  dogs  with  pancreatic  dia- 
betes, an  abundant  elimination  of  sugar  takes  place  even  on  the  complete 
exclusion  of  carbohydrates  from  the  food,  and  the  formation  of  .sugar  in 
these  cases  is  generally  considered  as  derived  from  the  protein  substances. 

Artificial  pancreatic  diabetes  may  also  in  other  respects  present  exactly 
the  same  picture  as  diabetes  in  man;  but  opinions  differ  as  to  the  cause  of 
this  diabetes.  According  to  the  Cavazzaxi  brothers,  as  well  as  Chauveau 
and  Kaufmaxn.3  pancreatic  diabetes  is  not  or  not  entirely  caused  by  a 

Weintraud  and  Laves,  Zeitschr.  f.  physiol.  Chem.,  19;  Haycraft,  ibid. ;  Minkowski, 
Arch.  f.  exp.  Path.  u.  Pharm.,  31. 

1  See  v.  Xoorden,  Die  Zuckerkrankheit,  3.  Aufl.,  1901. 

•  Minkowski,  Untersuchungen  iiber  Diabetes  mellitus  nach  Exstirpation  des 
Pankreas  (Leipzig,  1893);  v.  Noorden,  "Die  Zuckerkrankheit"  (Berlin,  1001),  which 
contains  a  very  copious  index  of  the  literature.  In  regard  to  diabetes  see  also  CI. 
Bernard,  Lemons  sur  le  diabete  (Paris),  and  Seegen,  Die  ^uckerbildung  in  Thierkorper 
{Berlin,  1S90). 

3  Cavazzani,  Centralbl.  f.  Physiol.,  7;  Chauveau  and  Kaufmann,  Mem.  soc.  biol., 
1893;  Kaufmann,  Arch,  de  Physiol,  (o),  7,  and  Conipt.  rend,  de  soc.  biol.,  47. 


25S  THE  LIVER. 

diminished  consumption  of  the  normal  quantity  of  sugar  formed,  but  to  an 
abnormally  increased  formation  of  sugar.  From  this  it  follows  that  the 
pancreatic  gland  has  a  regulating  action  on  the  formation  of  sugar  in  the 
liver,  a  retarding  action  which  is  caused  by  an  unknown  product  of  the 
internal  secretion  of  the  pancreas,  and  which  is  absent  on  the  extirpation  of 
the  gland.  Kaufmann  has  made  many  investigations  in  support  of  this 
view.  Among  other  things,  he  has  also  shown  that  on  the  extirpation  of 
the  pancreas  in  hyperglucaemic  animals  the  quantity  of  blood  is  quickly 
diminished  on  cutting  out  the  liver  or  the  portal  circulation.  Montuori  l 
has  arrived  at  similar  results,  since  the  large  quantity  of  sugar  in  the  blood 
of  dogs  on  ligaturing  the  pancreatic  vessels  was  diminished  on  subsequently 
ligaturing  the  liver  vessels.  Kausch  has  made  similar  observations  on 
birds  with  extirpated  pancreas  and  subsequent  liver  extirpation,  and 
Marcuse  2  has  likewise  shown  that  the  simultaneous  extirpation  of  the 
liver  and  pancreas  of  frogs  caused  no  glycosuria  in  any  case  (among  19), 
while  the  extirpation  of  the  pancreas  alone  in  12  animals  operated  upon 
(out  of  19)  caused  a  diabetes. 

There  remains  no  doubt  that  a  certain  relationship  exists  between  the 
liver  and  the  elimination  of  sugar  after  the  extirpation  of  the  pancreas, 
although  the  observations  do  not  lead  to  any  positive  conclusion.  The 
investigations  of  Minkowski,  Hedon,  Lancreaux,  Thiroloix,  and 
others  3  make  it  probable  that  special  chemical  products  of  the  internal 
secretion  of  the  pancreas  are  here  active.  According  to  these  investigations 
a  subcutaneously  transplanted  piece  of  the  gland  can  completely  perform 
the  functions  of  the  pancreas  as  to  the  sugar  exchange  and  the  sugar  elimi- 
nation, because  on  the  removal  of  the  intra-abdominal  piece  of  gland  the 
animal  in  this  case  does  not  become  diabetic.  But  if  the  subcutaneously 
imbedded  piece  of  pancreas  is  then  subsequently  removed,  an  active  elimina- 
tion of  sugar  appears  immediately. 

There  seems  lately  to  be  a  tendency  to  lean  towards  the  view  that  this 
internal  secretion  of  the  pancreas  is  related  to  the  so-called  islands  of 
Langerhans  of  the  pancreas.  No  one  is  acquainted  with  the  kind  of  sub- 
stance here  active.  The  glycolytic  activity  of  the  blood  shown  by  Lepine 
used  to  be  considered  by  him  as  due  to  a  glycolytic  enzyme  formed  in  the 
pancreas;  this  glycolysis  is  not  sufficient,  it  seems,  even  if  it  is  depend- 
ent upon  the  pancreas,  which  is  denied,  to  explain  the  transformation  in 
the  body  of  the  large  amount  of  sugar  present.  Perhaps  the  influence 
of  the  pancreas  must  be  sought  in  connection  with  the  muscles,  for,  as 

1  See  Maly's  Jahresber.,  26. 

2  Kausch,  Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Marcuse,  Du  Bois-Reymond 's  Arch., 
1894,  539. 

8  See  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  H6don,  Diabete  pancr6atique, 
Travaux  de  Physiologie  (Laboratoire  de  Montpellier,  1898). 


BILE  SECRETION.  259 

O.  Cohnheim  ■  has  shown,  one  can  obtain  a  cell-free  liquid  from  a  mixture  of 
muscle  and  pancreas  which  destroys  grape-sugar,  while  the  individual  organs 
alone  do  not  do  this. 

The  statement  of  Cohnhkim,  that  the  pancreatic  juice  and  muscle 
juice  together,  but  not  apart,  have  a  glycolytic  action,  has  been  disputed, 
as  Beveral  experimenters  such  as  Stoklasa  and  Czerny,  Simacek,  Fkix- 
BCHMTOT,  Arxheim  and  Rosenbaum  have  shown  a  glycolytic  property  of 
these  and  other  organs,  also  with  the  exclusion  of  bacteria.  The  precipi- 
tate produced  by  alcohol-ether  seems  to  be  more  active  than  the  press 
fluid  itself,  and  the  action  of  the  latter  was  often  very  weak.  To  those 
organs  which  contain  a  glycolytic  substance  also  belongs  the  liver,  in 
which  this  substance  is  absent  in  severe  cases  of  diabetes.  Cohnheim 's 
statements  have,  on  the  other  hand,  been  confirmed  by  Arnheim  and 
Rosenbaum  and  R.  Hirsch,  who  showed  that  the  pancreas  has  the 
•r  of  raising  the  glycolytic  property  of  the  liver  and  the  muscles  to  a 
considerable  degree.  The  pancreas  probably  acts  in  the  destruction  of 
the  sugar,  in  that  it  brings  about  the  action  of  the  glycolytic  enzymes 
present  in  the  other  organs  (Blumenthal).  Lepine,  who  has  already 
admitted  that  the  pancreas  has  a  direct  glycolytic  action  but  not  by  an 
internal  secretion,  is  also  of  the  opinion  that  the  glycolysis  produced  by  cell 
protoplasm  is  accelerated  by  the  pancreas.2 

The  Bile  and  its  Formation. 
C  By  the  establishment  of  a  biliary  fistula,  an  operation  which  was  first 
r  performed  by  Schwann  in  1844  and  which  has  been  improved  lately  by 
1  > a-tre  and  Pawlow,3  it  is  possible  to  study  the  secretion  of  the  bile.  This 
secretion  is  continuous,  but  witli  varying  intensity.  It  takes  place  under 
a  very  low  pressure;  therefore  an  apparently  unimportant  hindrance  in  the 
outflow  of  the  bile,  namely,  a  stoppage  of  mucus  in  the  exit,  or  the  secretion 
of  large  quantities  of  viscous  bile,  may  cause  stagnation  and  absorption  of, 
the  bile  by  means  of  the  lymphatic  vessels  (absorption  icterusj 

The  quantity  of  bile  secreted  in  the  twenty-four  hours  in  dogs  can  be 
exactly  determined.  The  quantity  secreted  by  different  animals  varies, 
and  the  limits  are  2.9-36.4  grams  of  bile  per  kilo  of  weight  in  the  twenty- 
four  hours.4 

1  Zeitschr.  f.  physiol.  Chem.,  39. 

:  Stoklasa,  Osterreich.  Chem.  Zeitung,  1903;  Czerny,  Ber.  d.  d.  chem.  Gesellsch., 
.'{(>;  -imacek,  Centralbl.  f.  Physiol.,  17;  Feinschmidt,  Hofmeister's  Beitriige,  4;  Arn- 
heim and  Rosenbaum,  Zeitschr.  f.  physiol.  Chem.,  40;  R.  Hirsch,  Hofmeister's  Bei- 
fcrfige,  4;  Blumenthal,  Deutsch.  med.  Wochenschr. ,  1903;  Lupine,  La  semaine  m6- 
dicale,  1903. 

'Schwann,  Arch.  f.  (Anat.  u.)  Physiol.,  1844;  Dastre,  Arch,  de  Physiol.  (5),  2; 
Pawlow,  Ergebnisse  der  Physiol.,  1,  Abt.  1. 

4  In  regard  to  the  quantity  of  bile  secreted  in  animals  see  Heidenhain,  Die  Gallenab- 


260  THE   LIVER. 

The  statements  as  to  the  extent  of  bile  secretion  in  man  are  few  and 
not  to  be  depended  on.  Ranke  found  (using  a  method  which  is  not  free 
from  criticism)  a  secretion  of  14  grams  of  bile  with  0.44  gram  of  solids  per 
kilo  in  twenty-four  hours.  Noel-Patox,  Mayo-Robsox,  Hammarstex, 
Pfaff  and  Balch,  and  Braxd  *  have  found  a  variation  between  514  and 
10S3  c.  c.  per  twenty-four  hours.  Such  determinations  are  of  doubtful  value, 
because  in  most  cases  it  follows  from  the  composition  of  the  collected  bile 
that  the  fluid  is  not  the  result  of  a  secretion  of  normal  liver  bile. 

The  quantity  of  bile  secreted  is,  however,  as  specially  shown  by  Stadel- 
maxx,2  subject  to  such  great  variation  even  under  physiological  conditions 
that  the  study  of  these  circumstances  which  influence  the  secretion  is  very 
difficult  and  uncertain.  The  contradictory  statements  by  different  investi- 
gators may  probably  be  explained  by  this  fact. 

In  starvation  the  secretion  diminishes.  According  to  Lukjaxow  and 
Albertoxi,3  under  these  conditions  the  absolute  quantity  of  solids  de- 
creases, while  the  relative  quantity  increases.  After jpartaking  of  food  the 
secretion  increases  again.  The  statements  are  very  contradictory  in  regard 
to  the  time  necessary  after  partaking  of  food  before  the  secretion  reaches 
its  maximum.  After  a  careful  examination  and  compilation  of  all  the 
existing  statements  Heidexhaix  *  has  come  to  the  conclusion  that  in  dogs 
the  curve  of  rapidity  of  secretion  shows  two  maxima,  the  first  at  the  third 
to  fifth  hour  and  the  second  at  the  thirteenth  to  fifteenth  hour,  after  par- 
taking of  food.  According  to  Barbera5  the  time  when  the  maximum 
occurs  is  dependent  upon  the  kind  of  food.  With  carbohydrate  food  it 
is  two  to  three  hours,  after  proteid  food  three  to  four  hours,  and  with  fat 
diet  it  is  five  to  seven  hours  after  feeding. 

According  to  the  older  statements,  the  proteids,  of  all  the  various  foods, 
cause  the  greatest  secretion  of  bile,  while  the  carbohydrates  diminish,  or 
at  least  excite  much  less,  than  the  proteids.  This  coincides  with  the  recent 
observations  of  Barbera.  The  authorities  are  by  no  means  agreed  as  to 
the  action  of  the  fats.  While  many  older  investigators  have  not  observed 
any  increase,  but  rather  the  reverse,  in  the  secretion  of  bile  after  feeding 
with  fats,  the  researches  of  Barbera  show  an  undoubted  increase  in  the 

sonderung,  in  Hermann's  Handbuch  der  Physiol.,  5,  and  Stadelmann,  Der  Icterus  und 
seine  verschiedenen  Formen  (Stuttgart,  1891). 

1  Ranke,  Die  Blutvertheilung  und  der  Thatigkeitsweohsel  der  Organe  (Leipzig, 
1871);  Noel-Paton,  Rep.  Lab.  Roy.  Coll.  Edinburgh,  3;  Mayo-Robson,  Proc.  Roy.  Soc, 
47;  Hammarsten,  Nova  act.  Reg.  Soc.  Scient.  Upsala  (3),  16;  Pfaff  and  Balch,  Journ. 
of  Exp.  Med.,  1897;  Brand,  Pfliiger's  Arch.,  90. 

2  Stadelmann,  Der  Icterus,  etc.     Stuttgart,  1891. 

3  Lukjanow,  Zeitschr.  f.  physiol.  Chem.,  1G;  Albertoni,  Recherches  sur  la  s6ct6- 
tion  biliaire.     Turin,  1893. 

*  Hermann's  Handb.,  5,  and  Stadelmann,  Der  Icterus,  etc. 

*  Centralbl.  f.  Physiol.,  12  and  16. 


THE  BILE.  261 

secretion  of  bile  on  fat  feeding,  greater  even  than  after  carbohydrate  feed- 
ing. According  to  Rosenberg  olive-oil  is  a  strong  cholagogue,  a  statement 
which,  according  to  other  investigators — Mandelstamm,  Doyon  and 
Dufourt  ' — is  not  sufficiently  proved. 

As  Barbera  has  shown,  a  close  relationship  exists  between  the  bile  secre- 
tion and  the  quantity  of  urea  formed,  as  an  increase  in  the  first  goes  hand 
in  hand  with  an  increase  of  the  latter.  The  bile  is,  therefore,  according  to 
him,  a  product  of  disassimilation,  whose  quantity  rises  and  falls  with  the 
degree  of  activity  of  the  liver. 

The  question  whether  there  exist  special  medicinal  bodies,  so-called 
cholagogues,  which  have  a  specific  excitant  action  on  the  secretion  of  bile 
has  been  answered  in  very  different  ways.  Many,  especially  the  older 
investigators,  have  observed  an  increase  in  the  bile  secretion  after  the  use 
of  certain  therapeutic  agents,  such  as  calomel,  rhubarb,  jalap,  turpentine, 
olive-oil,  etc.;  while  others,  especially  the  more  recent  investigators,  have 
arrived  at  quite  opposite  results.  From  all  appearances  this  contradiction 
is  due  to  the  great  irregularity  of  the  normal  secretion,  which  may  be  readily 
mistaken  in  tests  with  therapeutic  agents. 

/""""Schiff's  view,  that  the  bile  absorbed  from  the  intestinal  canal  increases 
the  secretion  of  bile  and  hence  acts  as  a  cholagogue,  seems  to  be  a  positively 
proven  fact  by  the  investigations  of  several  experimenters. vSodium 
salicylate  is  also  perhaps  a  cholagogue  (Stadelmann,  Doyon  and  Du- 
fourt). 
"  lrnebile  is  a  mixture  of  the  secretion  of  the  liver-cells  and  the  so-called 

'mucus  which  is  secreted  by  the  glands  of  the  biliary  passages  and  by  the 
mucous  membrane  of  the  gall-bladder.  The  secretion  of  the  liver,  which 
is  generally  poorer  in  solids  than  the  bile  from  the  gall-bladder,  is  thin  and 
clear,  while  the  bile  collected  in  the  gall-bladder  is  more  ropy  and  viscous 
on  account  of  the  absorption  of  water  and  the  admixture  of  "mucus,"  and  -1 
cloudy  because  of  the  admixture  of  cells,  pigments,  and  the  like.  The 
specific  gravity  of  the  bile  from  the  gall-blader  varies  considerably,  being 
in  man  between  1.010  and  1.040.  Its  reaction  is  alkaline  to  litmus.  The 
color  changes  in  different  animals;  golden-yellow,  yellowish-brown,  olive- 

1  Barbera,  Bull,  della  scienz.  med.  di  Bologna  (7),  5,  and  Maly's  Jahresber.,  24,  and 
Centralbl.  f.  Physiol.,  12  and  16;  Rosenberg,  Pfliiger's  Arch.,  46;  Mandelstamm,  Ueber 
den  Einfluss  einiger  Arzneimittel  auf  Sekretion  und  Zusammensetzung  der  Galle  (Dissert. 
Dorpat,  1890);  Doyon  and  Dufourt,  Arch,  de  Physiol.  (5),  9.  In  regard  to  the  action 
of  various  foods  on  the  secretion  of  bile  see  also  Heidenhain,  1.  c. ;  Stadelmann,  Der 
Icterus;  and  Barbera,  1.  c. ;  A.  Falloise,  Bull,  de  l'Acad.  Roy.  de  Belgique,  No.  S,  1903. 

3  Schiff ,  Pfliiger's  Arch.,  3.  See  Stadelmann,  Der  Icterus,  and  the  dissertations  of 
his  pupils,  especially  Winteler,  ' '  Experimentelle  Beitriige  zur  Frage  des  Kreislaufes 
der  Galle"  (Inaug.-Diss.  Dorpat,  1892),  and  Gartner,  "Experimentelle  Beitrage  zur 
Physiol,  und  Path,  der  Gallensekretion "  (Inaug.-Diss.  Jurjew,  1893);  also  Stadelmann, 
"Ueber  den  Kreislauf  der  Galle,"  Zeitschr.  f.  Biologie,  34. 


262  THE  LIVER. 

brown,  brownish-green,  grass-green,  or  bluish-green.  Bile  obtained  from 
an  executed  person  immediately  after  death  is  ordinarily  golden-yellow  or 
yellow  with  a  shade  of  brown.  Still  cases  occur  in  which  fresh  human  bile 
from  the  gall-bladder  has  a  green  color.  The  ordinary  post-mortem  bile 
has  a  variable  color.  The  bile  of  certain  animals  has  a  peculiar  odor;  as 
example,  ox-bile  has  an  odor  of  musk,  especially  on  warming.  The  taste 
of  bile  is  also  different  in  different  animals.  Human  as  well  as  ox-bile  has 
a  bitter  taste  with  a  sweetish  after-taste.  The  bile  of  the  pig  and  rabbit 
has  an  intensely  persistent  bitter  taste.  On  heating  bile  to  boiling  it  does 
not  coagulate.  It  contains  (in  the  ox)  only  traces  of  true  mucin,  and  its 
ropy  properties  depend,  it  seems,  chiefly  on  the  presence  of  a  nucleoalbumin 
similar  to  mucin  (Paijkull).  Hammarsten  *  has,  on  the  contrary,  found 
true  mucin  in  human  bile.  The  specific  constituents  of  the  bile  are  bile- 
acids  combined  with  alkalies,  bile-pigments,  and,  besides  small  quantities  of 
lecithin,  cholesterin,  soaps,  neutral  fats,  urea,  ethereal  sulphuric  acid,  traces 
of  conjugated  glucuronic  acids  and  mineral  substances,  chiefly  chlorides, 
besides  phosphates  of  calcium,  magnesium,  and  iron.  Traces  of  copper 
also  occur:___/^" 

-salts.  The  bile-acids  which  thus  far  have  best  been  studied  may 
be  divided  into  two  groups,  the  glycocholic  and  taurocholic  acid  groups.  As 
found  by  Hammarsten,2  a  third  group  of  bile-acids  occurs  in  the  shark  and 
probably  also  in  other  animals.  These  are  rich  in  sulphur,  and  like  the  ethe- 
real sulphuric  acids  they  split  off  sulphuric  aoid  on  boiling  with  hydro- 
chloric acid.  All  glycocholic  acids  contain  nitrogen,  but  are  free  from  sulphur 
and  can  be  split  with  the  addition  of  water  into  glycocoll  (aminoacetic  acid) 
and  a  nitrogen-free  acid,  cholic  acid.  All  taurocholic  acids  contain  nitrogen 
and  sulphur  and  are  split,  with  the  addition  of  water,  into  taurin  (amino- 
ethylsulphonic  acid)  and  cholic  acid.  The  reason  for  the  existence  of  differ- 
ent glycocholic  and  taurocholic  acids  depends  on  the  fact  that  there  are 
several  cholic  acids. 

The  conjugated  bile-acid  found  in  the  shark,  and  called  scymnol  sulphuric  acid 
by  Hammarsten,  yields  as  cleavage  products  sulphuric  acid  and  a  non-nitrogenous 
substance,  scymnol  (C^H^Os),  which  gives  the  characteristic  color  reactions  of 
cholic  acid. 


I  The  different  bile-acids  occur  in  the  bile  as  alkali  salts,  generally  in 
(combination  with  sodium/ and  also  in  sea-fishes,  although  this  is  contrary 
to  the  older  statements  (Zanetti3).  In  the  bile  of  certain  animals  we 
find  almost  solely  glycocholic  acid,  in  others  only  taurocholic  acid,  and 
in  other  animals  a  mixture  of  both  (see  further  on). 

1  Paijkull,  Zeitschr.  f.  physiol.  Chem.,  12;  Hammarsten,  1.  c,  Nova  Act.  (3),  16. 

2  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  24. 

3  See  Chem.  Centralis,  1003,  1,  180. 


BILE-SALTS.  263 

All  alkali  salts  of  the  biliary  acids  are  soluble  in  water  and  alcohol,  but 
insoluble  in  ether.  Their  solution  in  alcohol  is  therefore  precipitate  1  by 
ether,  and  this  precipitate,  with  proper  care  in  manipulation,  gives,  for 
nearly  all  kinds  pf  bile  thus  far  investigated,  rosettes  or  balls  of  fine  needles 
or  four-  to  six-sided  prisms  (Plattner's  crystallized  bile)J^/Fresh  human 
bile  also  crystallizes  readily.  The  bile-acids  and  their  salts  are  optically 
active  and  dextrorotatory.  The  former  are  dissolved  by  concentrated  sul- 
phuric acid  at  the  ordinary  temperature,  forming  a_xgddish-yellow  liquid 
which  has  a  beautiful  green  fluorescence.  /T)n  carefully  warming  with  con^N. 
centrated  sulphuric  acid  and  a  little  cane4ugar,  the  bile-acids  give  a  beauti- 
ful cherry-red  or  reddish-violet  liquid.     Pettenkofer 's  reaction  for  bile; / 

jicids  is  based  on  this  behavior .y  >^^ 

T^ettenkofer's  test  for  bile-acids  is  performed  as  follows :  A  small  \ 
quantity  of  bile  in  substance  is  dissolved  in  a  small  porcelain  dish  in  con- 
centrated sulphuric  acid  and  warmed,  or  some  of  the  liquid  containing  the 
bile-acids  is  mixed  with  concentrated  sulphuric  acid,  taking  special  care  in 
both  cases  that  the  temperature  does  not  rise  higher  than  60-70°  C.  Then 
a  10  per  cent  solution  of  cane-sugar  is  added,  drop  by  drop,  continually 
stirring  with  a  glass  rod.  The  presence  of  bile  is  indicated  by  the  produc- 
tion of  a  beautiful  red  liquid,  whose  color  does  not  disappear  at  the  ordinary 
temperature,  but  becomes  more  bluish  violet  in  the  course  of  a  day.  This 
red  liquid  shows  a  spectrum  with  two  absorption-bands,  the  one  at  F  and 
the  other  between  D  and  E,  near  E. 

This  extremely  delicate  test  fails,  however,  when  the  solution  is 
heated  too  high  or  if  an  improper  quantity — generally  too  much — of 
the  sugar  is  added.  In  the  last-mentioned  case  the  sugar  easily  car- 
bonizes and  the  test  becomes  brown  or  dark  brown.  The  reaction  fails 
if  the  sulphuric  acid  contains  sulphurous  acid  or  the  lower  oxides  of  nitro- 
gen. Many  other  substances,  such  as  proteids,  oleic  acid,  amyl  alcohol, 
and  morphine,  give  a  similar  reaction,  and  therefore  in  doubtful  cases 
the  spectroscopic  examination  of  the  red  solution  must  not  be  for- 
gotten. 

Pettenkofer 's  test  for  the  bile-acids  depends  essentially  on  the  fact 
that  furfurol  is  formed  from  the  sugar  by  the  sulphuric  acid,  and  this  body  ' 
can  therefore  be  substituted  for  the  sugar  in  this  test  (Mylius)./  Accord- 
ing to  "Mylius  and  v.  Udranszky1  a  1  p.  m.  solution  of  furTurol  should 
be  used.  Dissolve  the  bile,  which  must  first  be  purified  by  animal  charcoal, 
in  alcohol.  To  each  cubic  centimeter  of  alcoholic  solution  of  bile  in  a  test- 
tube  add  1  drop  of  the  furfurol  solution  and  1  c.  c.  concentrated  sul- 
phuric acid,  and  cool  when  necessary,  so  that  the  test  does  not  become  too 
warm.     This  reaction,  when  performed  as  described,  will  detect  -fa  to  -fa 

1  Mylius,  Zeitschr.  f.  physiol.  Chem.,  11;  v.  Udranszky,  ibid.,  12. 


264  THE  LIVER. 

milligram  cholic  acid  (v.  Udranszky).    Other  modifications  of  Petten- 
kofer's  test  have  been  proposed. 

Glycocholic  Acid.  The  constitution  of  the  glycocholic  acid,  occurring, 
in  human  and  ox-bile,  and  which  has  been  most  studied  is  represented  by 
the  formula  C26H43N06.  Glycocholic  acid  is  absent  or  nearly  so  in  the  bile 
of  carnivora.  On  boiling  with  acids  or  alkalies  this  acid,  which  is  analogous 
to  hippuric  acid,  is  converted  into  cholic  acid  and  glycocoll. 

Glycocholic  acid  crystallizes  in  fine,  colorless  needles  or  prisms.  It  is 
soluble  with  difficulty  in  water  (in  about  300  parts  cold  and  120  parts  boil- 
ing water),  and  is  easily  precipitated. from  its  alkali-salt  solution  by  the 
addition  of  dilute  mineral  acids.  It  is  readily  soluble  in  strong  alcohol,  but 
with  great  difficulty  in  ether.  The  solutions  have  a  bitter  but  at  the  same 
time  sweetish  taste.  The  acid  melts  at  138-140°  C.  (Medvedew  *) .  The 
salts  of  the  alkalies  and  alkaline  earths  are  soluble  in  alcohol  and  water; 
those  of  the  heavy  metals  are  mostly  insoluble  or  soluble  with  difficulty 
in  water.  The  solution  of  the  alkali  salts  in  water  is  precipitated  by  sugar 
of  lead,  cupric  and  ferric  salts,  and  silver  nitrate. 

Glycocholeic  Acid  is  a  second  glycocholic  acid,  first  isolated  by  Wahl- 
grex  2  from  ox-bile  and  has  the  formula  C26H43N05  or  C2?H45N05.  This 
acid,  which  on  hydrolytic  cleavage  yields  glycocoll  and  choleic  acid,  has 
also  been  detected  in  human  bile  and  the  bile  of  the  musk-ox  (Hammar- 
stex  3) . 

Glycocholeic  acid  may,  like  glycocholic  acid,  crystallize  in  tufts  of 
fine  needles,  but  is  often  obtained  as  short  thick  prisms.  It  is  much  more 
insoluble  in  water,  even  on  boiling,  than  glycocholic  acid,  and  it  melts  at 
175-176°  C.  The  alkali  salts  are  soluble  in  water  and  have  a  pure  bitter 
taste  and  are  more  readily  precipitated  by  neutral  salts  (NaCl)  than  the 
glycocholates.  The  solution  of  the  alkali  salts  is  not  only  precipitated 
by  the  salts  of  the  heavy  metals,  but  also  by  the  salts  of  barium,  calcium 
and  magnesium. 

The  preparation  of  the  pure  glycocholic  acids  may  be  performed  in,several 
ways.  The  bile,  which  has  been  freed  from  mucus  by  means  of  alcohol  and 
the  alcohol  removed  by  evaporation,  may  be  precipitated  by  a  solution  of 
lead  acetate.  The  precipitate  is  then  decomposed  by  a  soda  solution  and 
heat,  evaporated  to  dryness,  and  the  residue  extracted  with  alcohol,  which 
dissolves  the  alkali  glycocholate.  The  alcohol  is  distilled  from  the  filtered 
solution  and  the  residue  dissolved  in  water;  this  solution  is  now  decolorized 
by  animal  charcoal  and  the  glycocholic  acid  precipitated  from  the  solution 
by  the  addition  of  a  dilute  mineral  acid.  The  mixture  of  the  two  glyco- 
cholic acids  is  freed  from  mineral  acid  by  carefully  washing  with  water 

1  Centralbl.  f.  Physiol.,  14. 

7  Zeitschr.  f.  physiol.  Chem.,  36. 

8  Not  published. 


COMPOSITION  OF   THE   BILE.  275 

Older  and  less  complete  analyses  of  human  bile  have  been  made  by 
Frerichs  and  v.  Gorup-Besaxez.1  The  bile  analyzed  by  them  was  from 
perfectly  healthy  persons  who  had  been  executed  or  accidentally  killed. 
The  two  analyses  of  Frerichs  are,  respectively,  of  (I)  an  lS-y  ear-old  and 
(II)  a  22-year-old  male.  The  analyses  of  v.  Gorup-Besaxez  are  of  (I)  a 
man  of  49  and  (II)  a  woman  of  29.  The  results  are,  as  usual,  in  parts 
per  1000. 

Frerichs.  v.  Gorcp-Besanez. 

I.  II.  I.  II. 

Water 860.0  859.2  822.7  898.1 

Solids 140.0  140.8  177.3  101.9 

Biliary  salts 72.2  91.4  107.9  56.5 

Mucus  and  pigments 26.6  29.8  22.1  14.5 

Cholesterin 1.6  2 . 6  |  <-  q  on  q 

Fat 3.2  9.2  f  4'   6  "*Uy 

Inorganic  substances 6.5  7.7  10.8  6.2 

Human  liver-bile  is  poorer  in  solids  than  the  bladder-bile.  In  several 
cases  it  contained  only  12-18  p.  m.  solids,  but  the  bile  in  these  cases  is 
hardly  to  be  considered  as  normal.  Jacobsex  found  22.4-22.8  p.  m. 
solids  in  a  specimen  of  bile.  Hammarstex,  who  had  occasion  to  analyze 
the  liver-bile  in  seven  cases  of  biliary  fistula,  has  repeatedly  found  25-28 
p.  m.  solids.  In  a  case  of  a  corpulent  woman  the  quantity  of  solids  in  the 
liver-bile  varied  between  30.10-36.8  p.  m.  in  ten  days.  Brand2  has  ob- 
served still  higher  figures,  more  than  40  p.  m.  in  a  couple  of  cases.  This 
investigator  suggests  that  the  bile  from  an  imperfect  fistula,  when  it  is 
partly  absorbed,  is  richer  in  solids  than  when  it  comes  from  a  perfect  fistula. 

The  molecular  concentration  of  human  bile,  according  to  Braxd, 
Boxaxi,  and  Strauss3  is  nearly  always  identical  with  that  of  the  blood, 
although  the  amount  of  water  and  solids  varies.  The  freezing-point  varies 
only  between  —0.54°  and  —0.58°.  This  stability  of  the  osmotic  pressure 
h  explained  by  the  fact  that  in  concentrated  biles  with  larger  amounts  of' 
organic  substances  (with  larger  molecules)  the  amount  of  inorganic  salts 
Is  lower.4 

Hurr|an  bile  sometimes,  but  not  always,  contains  sulphur  in  an  ethereal 
sulphuric-acid-like  combination.  The  quantity  of  such  sulphur  may  even 
amount  to  £-■$•  of  the  total  sulphur.  Human  bile  is  habitually  richer  in 
glycocholic  than  in  taurocholic  acid.  In  six  cases  of  liver-bile  analyzed  by 
Hammarstex  the  "relationship  of  taurocholic  to  glycocholic   acid  varied 

1  See  Hoppe-Seyler,  Physiol.  Chem.,  301;  Socoloff,  Pfliiger's  Arch..  12:  Trifanow- 
ski,  ibid.,  9;   Frerichs  in  Hoppe-Seyler 's  Physiol.  Chem..  290:   v.  Gorup-Besanez,  ibid. 

2Jacobsen,  Ber.  d.  deutsch.  chem.  Gesellsch.,  6;  Hammarsten,  Nova  Acta  Reg. 
Soc.  Scient.  Upsala,  16;   Brand,  Pfliiger's  Arch.,  90. 

3  Brand,  1.  c. ;  Bonani,  Biochem.  Centralbl.,  1;  Strauss,  Berl.  klin.  Wochensehr., 
1903. 

4  See  Brand,  1.  c. ;  Hammarsten,  1.  c. 


276  THE  LIVER. 

between  1 : 2.07  and  1 : 1-1.36.     The  bile  analyzed  by  Jacobsen  contained 
no  taurocholic  acid. 

As  an  example  of  the  composition  of  human  liver-bile  the  following 
results  of  three  analyses  made  by  Hammarsten  are  given.  The  results 
are  calculated  in  parts  per  1000. * 

Solids 25.200  35.260  25.400 

Water 974.800  964.740  974.600 

Mucin  and  pigments 5 .  290  4 .  290  5 .  150 

Bile-salts 9.310  18.240  9.040 

Taurocholate 3 .  034  2 .  079  2 .  180 

Glvcocholate 6.276  16.161  6.860 

Fattv  acids  from  soaps 1 .  230  1 .  360  1 .  010 

Chofesterin 0.630  1 .600  1 .500 

Lecithin )    n  99n  0 .  475  0 .  650 

Fat J    U^U  0.956  0.610 

Soluble  salts 8.070  6.760  7.250 

Insoluble  salts 0.250  0.490  0.210 

Amongst  the  mineral  constituents  the  chlorine  and  sodium  occur  to  the 
greatest  extent.  The  relationship  between  potassium  and  sodium  varies 
considerably  in  different  samples.  Sulphuric  acid  and  phosphoric  acid  occur 
only  in  very  small  quantities. 

Bagixsky  and  Sommerfeld  2  have  found  true  mucin,  mixed  with 
some  nucleoalbumin,  in  the  bladder-bile  of  children.  The  bile  contained 
on  an  average  896.5  p.  m.  water;  103.5  p.  m.  solids;  20  p.  m.  mucin;  9.1 
p.  m.  mineral  substances;  25.2  p.  m.  bile-salts  (of  which  16.3  p.  m.  were 
glvcocholate  and  8.9  p.  m.  taurocholate);  3.4  p.  m.  cholesterin;  6.7  p.  m. 
fat,  and  2.8  p.  m.  leucin.3 

The  quantity  of  pigment  in  human  bile  is,  according  to  Noel-Paton, 
0.4-1.3  p.  m.  for  a  case  of  biliary  fistula.  The  method  used  in  determining 
the  pigments  in  this  case  was  not  quite  trustworthy.  More  exact  results 
.obtained  by  spectrophotometric  methods  are  on  record  for  dog-bile. 
According  to  Stadelmann  *  dog-bile  contains  on  an  average  0.6-0.7  p.  m. 
bilirubin.  At  the  most  only  7  milligrams  of  pigment  are  secreted  per  kilo 
of  body  in  the  twenty-four  hours. 

In  animals  the  relative  proportion  of  the  two  acids  varies  considerably. 
It  has  been  found,  on  determining  the  amount  of  sulphur,  that,  so  far  as 
the  experiments  have  gone,  taurocholic  acid  is  the  prevailing  acid  in  car- 
nivorous mammals,  birds,  snakes,  and  fishes.  Among  the  herbivora,  sheep 
and  goats  have  a  predominance  of  taurocholic  acid  in  the  bile.  Ox-bile 
sometimes  contains  taurocholic  acid  in  excess,  in  other  cases  glycocholic  acid 

1  Recent  quantitative  analyses  may  be  found  in  Brand,  1.  c;  v.  Zeynek,  Wien. 
klin.  "Wochenschr.,  1899;   Bonani,  1.  c. 

2  Verhandl.  d.  physiol.  Gesellsch.  zu  Berlin,  1894-95. 

8  Analyses  of  bile  from  children  may  be  found  in  Heptner,  Maly's  Jahresber.,  30. 
*  Noel-Paton,  Rep.  Lab.  Roy.  Soc.  Coll.  Phys.  Edinburgh,  3;    Stadelmann,  Der 
Icterus. 


CHOLEIC  ACID.  267 

The  alkali  Balta  are  readily  soluble  in  water,  but  when  treated  with  a 
concentrated  caustic  or  carbonated  alkali  solution  may  be  separated  as  an 
oily  mass  which  becoi  alline  on  cooling.     The  alkali  salts  are  not 

readily  soluble  in  alcohol,  and  on  the  evaporation  of  the  alcohol  they  may 
crystallize.  The  specific  rotatory  power  of  the  sodium  -alt  is  (a)\)=  -f 
31. 4°. *  The  watery  solution  of  the  alkali  salts,  when  not  too  dilute,  is 
precipitated  immediately  or  after  some  time  by  sugar  of  lead  or  by  barium 
chloride.  The  barium  salt  crystallizes  in  fine,  silky  needles,  and  it  is  rather 
insoluble  in  cold  but  somewhat  easily  soluble  in  warm  water.  The  barium 
salt,  as  well  as  the  lead  salt  which  is  insoluble  in  water,  is  soluble  in  warm 
alcohol . 

Choleic  Acid  (C,5H42(  >4.  Latschixoff)  is  another  cholic  acid  which, 
according  to  Lass.yr-Cohx  3  has  the  formula  C^H^O^  This  acid,  which 
occurs  in  varying  but  always  small  quantities  in  ox-bile,  yields  dehydro- 
choleic  acid,  C,4H3404,  and  then  cholanic  acid,  C^H^Og,  and  isocholanic  acid 
on  oxidation. 

Choleic  acid  crystallizes  when  free  from  water  in  hexagonal,  vitreous 
prisms  with  pointed  ends,  melting  at  l.s5-190°  C.  The  crystalline  acid  con- 
taining water  melts  at  135-140°  C.  (Lat-chixoff).  The  acid  dissolves  in 
water  with  difficulty  and  is  also  relatively  difficultly  soluble  in  alcohol.  It 
has  an  intense  bitter  taste  and  gives  the  Mylius  iodine  reaction  for  cholic 
acid.  The  specific  rotation  is  («)d=  +48.87°  (Vahlen)-  The  barium 
salt  which  crystallizes  from  the  hot  alcoholic  solution  as  spherical  aggre- 
gations of  radial  needles  is  more  difficultly  soluble  in  water  than  the 
jponding  cholate. 

The  relation  of  choleic  acid  to  ilcsnxi/cholic  acid  is  not  known.  Accord- 
ing to  Latschixoff  and  Lassar-Cohn  both  acids  are  identical,  and  the  fact  that 
the  desoxycholic  acid  on  oxidation  uho  yields  dehydrocholeic  acid  and  cholanic 
acid  seems  to  prove  this  (Pbegl).  Desoxycholic  acid  i*,  on  the  contrarv.  readily 
soluble  in  alcohol  and  has  a  lower  melting-point,  namely  172-173°  C,  when  free 
from  water  I  Pregl).  For  this  reason  Pregl  3  questions  whether  these  acids  are 
identical  or  are  isomeric  substances. 

Both  cholic  acids  are  best  prepared  from  ox-bile  which  has  been  boiled 
for  twenty-four  hours  with  baryta-water  or  caustic  soda.  Accordi 
Mylius,  '  boil  the  bile  for  twenty-four  hours  with  five  parts  its  weight  of  a  30 
per  cent  caustic-soda  solution,  replacing  the  water  lost  by  evaporation.  Now 
saturate  the  liquid  with  C02  and  evaporate  nearly  to  dryness.  The  residue 
is  extracted  with  9G  per  cent  alcohol  and  this  alcoholic  extract  diluted 
with  water  until  it  contains  at  the  most  20  per  cent  alcohol ;  it  is  then  com- 

1  >ee  Vahlen,  Zeitschr.  f.  physiol.  Chem.,  21. 

:  Latschinoff.  Ber.  d.  deutsch.  chem.  Gesellsch.,  IS  and  20:  Lassar-Cohn,  Aid.,  2»'.. 
and  Zeitschr.  f.  physiol.  Chem..  17.     See  also  Vahlen,  Zeitschr.  f.  physiol.  Chem..  83. 

MYien.  Sitz.-Ber.,  Ill;  Math.  Xatunv.  Kl..  1902;  Latschinoff,  1.  e. ;  Lassar-Cohn, 
1.  c.     See  also  Mylius,  Ber.  d.  d.  chem.  Gesellsch.,  19. 

4  Zeitschr.  f.  physiol.  Chem.,  12. 


26S  THE  LIVER. 

pletely  precipitated  with  a  BaCl2  solution.  The  precipitate,  which  contains 
besides  fatty  acids  also  the  choleic  acid,  is  filtered  and  the  cholic  acid  pre- 
cipitated from  the  filtrate  by  hydrochloric  acid.  After  the  cholic  acid  has 
gradually  crystallized  out  it  is  repeatedly  recrystallized  from  alcohol  or 
methyl  alcohol. 

Choleic  acid  may  be  obtained  from  the  above-mentioned  barium  pre- 
cipitate by  first  converting  the  barium  salt  into  sodium  salt  by  sodium  car- 
bonate and  then  fractionally  precipitating  the  fatty  acids  by  barium  acetate 
and  separating  the  choleic  acid  from  the  filtrate  by  hydrochloric  acid  and 
recrystallizing  several  times  from  glacial  acetic  acid. 

Pregl  1  has  suggested  a  somewhat  different  but  simpler  method  for 
preparing  cholic  acid  and  obtaining  the  desoxycholic  acid  from  ox-bile.  In 
regard  to  this  as  well  as  other  methods  of  preparation  we  must  refer  to 
the  original  communications  and  to  other  handbooks. 

Fellic  Acid,  C23H40O4,  is  a  cholic  acid,  so  called  by  Schotten,  which 
he  obtained  from  human  bile,  along  with  the  ordinary  acid.  This  acid  is 
crystalline,  is  insoluble  in  water,  and  yields  barium  and  magnesium  salts 
which  are  very  insoluble.  It  does  not  respond  to  Pettenkofer's  reaction 
easily  and  gives  a  more  reddish-blue  color. 

The  conjugate  acids  of  human  bile  have  not  been  sufficiently  investi- 
gated. To  all  appearance  human  bile  contains  under  different  circum- 
stances various  conjugate  bile-acids.  In  some  cases  the  bile-salts  of  human 
bile  are  precipitated  by  BaCl2  and  in  others  not.  According  to  the  latest 
statements  of  Lassar-Cohn  2  three  cholic  acids  may  be  prepared  from 
human  bile,  namely,  ordinary  cholic  acid,  choleic  acid,  and  fellic  acid. 

Lithofellic  Acid,  C20H36O4,  is  the  acid  related  to  cholic  acid  which  occurs  in 
the  oriental  bezoar  stones,  which  is  insoluble  in  water,  comparatively  easily  solu- 
ble in  alcohol,  but  only  slightly  soluble  in  ether.3 

The  hyo-glycocholic  and  cheno-taurocholic  acids,  as  well  as  the  glyco- 
cholic  acid  of  the  bile  of  rodents,  yield  corresponding  cholic  acids.  In  the 
polar  bear  a  third  cholic  acid  exists  besides  cholic  and  choleic  acids.  It 
is  called  ursocholeic  acid,  CleH30O4  or  C^H^C^  (Hammarsten  4) .  The  bile 
of  other  animals  (walrus,  sea-dog)  contains  special  cholic  acids  (Hammar- 

STENji*- — ' 

(On  boiling  with  acids,  on  putrefaction  in  the  intestine,  or  on  heating, 
cholic  acids  lose  water  and  are  converted  into  anhydrides,  the  so-called 
dyslysins.    The  dyslysin,  C24H3e03,  corresponding  to  ordinary  cholic  acid, 

1  L.  c. ,  Wien.  Sitzber. 

1  Schotten,  Zeitschr.  f.  physiol.  Chem.,  11;  Lassar-Cohn,  Ber.  d.  deutsch.  chem. 
Geseilsch.,  27. 

3  See  Jiinger  and  Klages,  Ber.  d.  deutsch.  chem.  Geseilsch.,  28  (older  literature). 

*  Zeitschr.  f.  physiol.  Chem.,  36. 

$  Not  published.  i 


BILE-PIGMENTS.  269 

which  occurs  in  freces,  is  amorphous,  insoluble  in  water  and  alkalies. 
Choloidic  acid,  C^H^O^  is  called  the  first  anhydride  or  an  intermediary 
product  in  the  formation  of  dyslysin.  On  boiling  dyslysins  with  caustic 
alkali  they  are  reconverted  into  the  corresponding  cholic  acid. 

The  Detection  of  Bile-acids  in  Animal  Fluids.  To  obtain  the 
bile-acids  pure  so  that  Pettenkofer's  test  can  be  applied  to  them,  the 
proteid  and  fat  must  first  be  removed.  The  proteid  is  removed  by  making 
the  liquid  first  neutral  and  then  adding  a  great  excess  of  alcohol,  so  that 
the  mixture  contains  at  least  85  vols,  per  cent  of  water-free  alcohol.  Now 
filter,  extract  the  precipitated  proteid  with  fresh  alcohol,  unite  all  filtrates, 
distill  the  alcohol,  and  evaporate  to  dryness.  The  residue  is  completely 
exhausted  with  strong  alcohol,  filtered,  and  the  alcohol  entirely  evaporated 
from  the  filtrate.  The  new  residue  is  dissolved  in  water,  and  filtered  if 
necessary,  and  the  solution  precipitated  by  basic  lead  acetate  and  am- 
monia. The  washed  precipitate  is  dissolved  in  boiling  alcohol,  filtered  while 
warm,  and  a  few  drops  of  soda  solution  added.  Then  evaporate  to  dryness, 
extract  the  residue  with  absolute  alcohol,  filter,  and  add  an  excess  of  ether. 
The  precipitate  now  formed  may  be  used  for  Pettenkofer's  test.  It  is 
not  necessary  to  wait  for  a  crystallization;  but  one  must  not  consider  the 
crystals  which  form  in  the  liquid  as  being  positively  crystallized  bile.  It  is 
also  possible  for  needles  of  alkali  acetate  to  be  formed.  For  the  detection 
of  bile-acids  in  urine  see  Chapter  XV. 

Bile-pigments.  The  bile-coloring  matters  known  thus  far  are  relatively 
numerous,  and  in  all  probability  there  are  still  more.  Most  of  the  known 
bile-pigments  are  not  found  in  the  normal  bile,  but  occur  either  in  post- 
mortem bile  or  principally  in  the  bile  concrements.  The  pigments  which 
occur  under  physiological  conditions  are  the  reddish-yellow  bilirubin,  the 
green  biliverdin,  and  sometimes  also  urobilin  or  a  closely  related  pigment. 
The  pigments  found  in  gall-stones  are  (besides  the  bilirubin  and  biliverdin) 
bilifuscin,  biliprasin,  bilihumin,  bilicyanin  (and  choletelin?) .  Besides  these, 
others  have  been  noticed  by  various  observers  in  human  and  animal  bile. 
The  two  above-mentioned  physiological  pigments,  bilirubin  and  biliverdin, 
are  those  which  serve  to  give  the  golden-yellow  or  orange-yellow  or  some- 
times greenish  color  to  the  bile,  or  when,  as  is  most  frequently  the  case  in 
ox-bile,  the  two  pgiments  are  present  in  the  bile  at  the  same  time,  pro- 
ducing the  different  shades  between  reddish-brown  and  green. 

Bilirubin.  This  pigment,  according  to  the  common  acceptation,  has  the 
formula  C18H18N203  (Malt)  and  is  designated  by  the  names  cholepyrrhin, 
bilipHjEin,  bilifulvin,  and  h^ematoidin.  It  occurs  chiefly  in  the  gall-stones 
as  liilirubin-calcium.  Bilirubin  is  present  in  the  liver-bile  of  all  vertebrates, 
and  in  the  bladder-bile  especially  in  man  andcarnivora;  sometimes,  however, 
the  latter  when  fasting  or  in  a  starving  condition  may  have  a  green  bile. 
It  occurs  also  in  the  contents  of  the  small  intestine,  in  the  blood-serum  of  the 
horse,  in  old  blood  extravasations  (as  hsematoidin) ,  and  in  the  urine  and 


270  THE  LIVER. 

the  yellow-colored  tissue  in  icterus.  It  is  converted  into  hydrobilirubin, 
C32H40N4O7  (Maly),  by  hydrogen  in  a  nascent  state,  and  then  shows  great 
similarity  to  the  urinary  pigment,  urobilin,  as  well  as  to  stercobilin  found  in 
the  contents  of  the  intestine  (Masius  and  Vanlair  *).  On  careful  oxida- 
tion bilirubin  yields  biliverdin  and  other  coloring-matters  (see  below). 

Bilirubin  is  derived  from  the  blood-pigment.  It  has  the  same  percentage 
composition  as  hsematoporphyrin  and  like  hsematin  it  yields  hsematinic 
acid  imide  as  an  oxidation  product  (Kuster  2). 

Bilirubin  is  partly  amorphous  and  partly  crystalline.  The  amorphous 
bilirubin  is  a  reddish-yellow  powder  of  nearly  the  same  color  as  amorphous 
antimony  sulphide;  the  crystalline  bilirubin  has  nearly  the  same  color  as 
crystallized  chromic  acid.  The  crystals,  which  can  easily  be  obtained  by 
allowing  a  solution  of  bilirubin  in  chloroform  to  evaporate  spontaneously, 
are  reddish-yellow,  rhombic  plates,  whose  obtuse  angles  are  often  rounded. 
On  crystallizing  from  hot  dimethylaniline  it  forms  on  cooling  broad  columns 
with  both  ends  sharply  cut. 

/  Bilirubin  is  insoluble  in  water  and  occurs  in  animal  fluids  as  soluble^ 
Cbihrubm-aJkalL/^Tt  is  very  slightly  soluble  in  ether,  benzene,  carbon 
disulphide,  amyl  alcohol,  fatty  oils,  and  glycerine.  It  is  somewhat  more 
soluble  in  alcohol.  In  cold  chloroform  it  dissolves  in  the  proportion  of 
1  :"600  and  is  much  more  readily  soluble  in  warm  chloroform.  In  cold  di- 
methylaniline it  dissolves  in  the  proportion  of  1 :  100,  and  in  hot  dimethyl- 
aniline  much  more  readily.  Its  solutions  show  no  absorption-bands, 
but  only  a  continuous  absorption  from  the  red  to  the  violet  end  of  the 
spectrum,  and  they  have,  even  on  diluting  greatly  (1:500000),  in  a  layer 
1.5  c.  m.  thick  a  decided  yellow  color.  If  a  dilute  solution  of  bilirubin- 
alkali  in  water  is  treated  with  an  excess  of  ammonia  and  then  with  a  zinc- 
chloride  solution,  the  liquid  is  first  colored  deep  orange  and  then  gradually 
olive-brown  and  then  green.  This  solution  first  gives  a  darkening  of  the 
violet  and  blue  part  of  the  spectrum  and  then  the  bands  of  alkaline  chole- 
cyanin  (see  below) ,  or  at  least  the  bands  of  this  pigment  in  the  red  between 
C  and  D  close  to  C.  This  is  a  good  reaction  for  bilirubin.  The  combina- 
tions of  bilirubin  with  alkalies  are  insoluble  in  chloroform,  and  bilirubin 
may  be  separated  from  its  solution  in  chloroform  by  shaking  with  dilute 
caustic  alkali  (differing  from  lutein).  Solutions  of  bilirubin-alkali  in  water 
are  precipitated  by  the  soluble  salts  of  the  alkaline  earths  and  also  by 
metallic  salts. 

/  If  an  alkaline  solution  of  bilirubin  be  allowed  to  stand  in  contact  with\ 
/  the  air,  it  gradually  absorbs  oxygen  and  green  biliverdin  is  formed.  This/' 
y  process  is  accelerated  by  warming.J^^ccording  to  Kuster  in  this  case 

1  Maly's  Wien.  Sitzungsber.,  57,  and  Annal.  d.  Chenx,  163;  Masius  and  Vanlair, 
Centralbl.  f.  d.  med.  Wissensch.,  1871,  369. 

2  Zeitschr.  f.  physiol.  Chem.,  30  and  35. 


BILIRUBIN  REACTIONS.  271 

the  alkali  also  has  a  splitting  action  upon  the  pigment  and  not  one  body  but 
several  arc  funned.  BiUveidin  18  also  formed  from  bilirubin  by  oxidation 
under  other  conditions.  A  green  coloring-matter  similar  in  appearance  is 
formed  by  the  action  of  other  reagents  such  as  CI,  Br,  and  I.  According  to 
JoLLES  l  by  the  use  of  Hubl's  iodine  solution  biliverdin  is  produced,  while 
according  to  others  (Thudichum,  Maly  2)  substitution  products  of  bili- 
rubin are  formed. 

Gmelin's  Reaction  for  Bile-pigments.  If  one  carefully  pours  under  a  ' 
solution  of  bilirubin-alkali  in  water  nitric  acid  containing  some  nitrous  acid, 
thi  re  is  obtained  a  series  of  colored  layers  at  the  juncture  of  the  two  liquids 
in  the  following  order  from  above  downwards:  green,  blue,  violet,  red,  and 
reddish-yellow.  This  color  reaction,  Gmelin's  test,  is  very  delicate  and 
serves  to  detect  the  presence  of  one  part  bilirubin  in  80,000  parts  liquid. 
The  green  ring  must  never  be  absent;  and  also  the  reddish-violet  must  be 
at  at  the  same  time,  otherwise  the' reaction  may  be  confused  with  that 
for  lutein,  which  gives  a  blue  or  greenish  ring.  The  nitric  acid  must  not 
contain  too  much  nitrous  acid,  for  then  the  reaction  takes  place  too  quickly 
and  it  does  not  become  typical.  Alcohol  must  not  be  present  in  the  liquid, 
because,  as  is  well  known,  it  gives  a  play  of  colors,  in  green  or  blue,  with 
the  acid. 

Hammarsten's  Reaction.  An  acid  is  first  prepared  consisting  of  1  vol. 
nitric  acid  and  19  vols,  hydrochloric  acid  (each  acid  being  about  25  per  cent). 
One  volume  of  this  acid  mixture,  which  can  be  kept  for  at  least  a  year,  is, 
when  it  has  become  yellow  by  standing,  mixed  with  4  vols,  alcohol.  If  a 
drop  of  bilirubin  solution  is  added  to  a  few  cubic  centimeters  of  this  color- 
less mixture  a  permanent  beautiful  green  color  is  obtained  immediately. 
On  the  further  addition  of  the  acid  mixture  to  the  green  liquid  all  the  colors 
of  Gmelin's  scale,  as  far  as  choletelin,  can  be  produced  consecutively. 
(  Huppert's  Reaction.  If  a  solution  of  bilirubin-alkali  Is  treated" with ^N 
milk  of  lime  or  with  calcium  chloride  and  ammonia,  a  precipitate  is  pro- 
duced consisting  of  bilirubin-calcium.  If  this  moist  precipitate,  which  has 
been  washed  with  water,  is  placed  in  a  test-tube  and  the  tube  half  filled  with 
alcohol  which  has  been  acidified  with  hydrochloric  acid,  and  heated  to  boiling 
for  some  time,  the  liquid  becomes  emerald-green  or  bluish-green  in  color. 

In  regard  to  the  modifications  of  <  Imelin's  test  and  certain  other  reac- 
tions for  bile-pigments,  see  Chapter  XV  (Urine). 

That  thecharacteristic  plav  of  colors  in  Gmelin's  test  is  the  result  of 
.an  oxidation  is  generally  admitted.  The  first  oxidation  step  is  the  green 
biliverdin.      Then    follows    a    blue   coloring-matter   which   Heinsius    and 

1  Kiister,  Ber.  d.  d.  chem.  Gesellsch.,  35;  Jolles,  Journ.  f.  prakt.  Chem.  (N.  F.),  59, 
avid  Pfluger's  Arch.,  7.">. 

'Thudichum,  Journ.  of  Chem.  Soc,  (2),  13,  and  Journ.  f.  prakt.  Chem.  (N.  F.), 
.>3;  Maly,  Wien.  Sitzungsber.,  72. 


272  THE  LIVER. 

Campbell  call  bilicyanin  and  Stokvis  calls  cholecyanin,  and  which  shows  a 
characteristic  absorption-spectrum.  The  neutral  solutions  of  this  coloring- 
matter  are,  according  to  Stokvis,  bluish-green  or  steel-blue  with  a  beautiful 
blue  fluorescence.  The  alkaline  solutions  are  green  and  have  no  marked 
fluorescence,  and  show  three  absorption-bands,  one  sharp  and  dark 
in  the  red  between  C  and  D,  nearer  to  C;  a  second,  less  well  defined, 
covering  D;  and  a  third,  between  E  and  F,  near  E.  The  strongly  acid 
solutions  are  violet-blue  and  show  two  bands,  described  by  Jaffe,  between 
the  lines  C  and  E,  separated  from  each  other  by  a  narrow  space  near  D.  A 
third  band  between  b  and  F  is  seen  with  difficulty.  The  next  oxidation  step 
after  these  blue  coloring-matters  is  a  red  pigment,  and  lastly  a  yellowish- 
brown  pigment,  called  choletelin  by  Maly,  which  in  neutral  alcoholic 
solutions  does  not  give  any  absorption  spectrum,  but  in  acid  solution  gives 
a  band  between  b  and  F.  On  oxidizing  cholecyanin  with  lead  peroxide, 
Stokvis  *  obtained  a  product  which  he  calls  choletelin,  which  is  quite  sim- 
ilar to  urinary  urobilin,  to  be  discussed  later. 

Bilirubin  is  best  prepared  from  gall-stones  of  oxen,  these  concretions 
being  very  rich  in  bilirubin-calcium.  The  finely  powdered  concrement  is 
first  exhausted  with  ether  and  then  with  boiling  water,  so  as  to  remove  the 
cholesterin  and  bile-acids.  The  powder  is  then  treated  with  hydrochloric 
acid,  which  sets  free  the  pigment;  washed  thoroughly  with  water  and  alcohol, 
dried,  and  extracted  repeatedly  with  boiling  chloroform.  The  bilirubin 
separates  from  the  chloroform  as  crusts,  which  are  treated  once  or  twice  in 
the  above  manner.  It  is  then  extracted  with  alcohol  and  precipitated  from 
its  chloroform  solution  by  alcohol  or  crystallized  from  dimethylaniline. 
The  crusts  of  bilirubin  which  separate  from  the  chloroform  solution  con- 
tain, according  to  Kuster,2  a  pigment  related  to  bilirubin,  poorer  in  nitrogen 
and  also  precipitable  by  alcohol.  The  quantitative  estimation  of  bilirubin 
may  be  made  by  the  spectro-photometrical  method,  according  to  the  steps 
suggested  for  the  blood-coloring  matters. 

Biliverdin,  C16H18N204.  This  body,  which  is  formed  by  the  oxidation  of 
bilirubin,  occurs  in  the  bile  of  many  animals,  in  vomited  matter,  in  the 
placenta  of  the  bitch  (?),  in  the  shells  of  birds'  eggs,  in  the  urine  in  icterus, 
and  sometimes  in  gall-stones,  although  in  very  small  quantities. 

Biliverdin  is  amorphous;  at  least  it  has  not  been  obtained  in  well- 
defined  crystals.  It  is  insoluble  in  water,  ether,  and  chloroform  (this  is 
true  at  least  for  the  artificially  prepared  biliverdin),  but  is  soluble  in  alcohol 
or  glacial  acetic  acid,  showing  a  beautiful  green  color.  It  is  dissolved  by 
alkalies,  giving  a  brownish-green  color,  and  this  solution  is  precipitated  by 
acids,  as  well  as  by  calcium,  barium,  and  lead  salts.     Biliverdin  gives 

1  Heinsius  and  Campbell,  Pfliiger's  Arch.,  4;  Stokvis,  Centralbl.  f.  d.  med.  Wis- 
sensch.,  1872,  785;  ibid.,  1873,  211  and  449;  Jaffe\  ibid.,  1868;  Maly,  Wien.  Sitzungs- 
ber.,  59. 

1  Ber  d.  d.  chem.  Gesellsch.,  35. 


BILIVERDIN  AND  OTHER  BILE-PIGMENTS.  273 

Huppert's,  Gmelin's,  and  Hammarsten's  reactions,  commencing  with 
the  blue  color.  It  is  converted  into  hydrobilirubin  by  nascent  hydrogen. 
On  allowing  the  green  bile  to  stand,  also  by  the  action  of  ammonium  sul- 
phide, the  biliverdin  may  be  reduced  to  bilirubin  (Haycraft  and  Scofield  '). 

Biliverdin  Is  most  simply  prepared  by  allowing  a  thin  layer  of  an  alkaline 
solution  of  bilirubin  to  stand  exposed  to  the  air  in  a  dish  until  the  color  is 
brownish-green.  The  solution  is  then  precipitated  by  hydrochloric  acid, 
the  precipitate  washed  with  water  until  no  HC1  reaction  is  obtained,  then 
dissolved  in  alcohol  and  the  pigment  again  separated  by  the  addition  of 
water.  Any  bilirubin  present  may  be  removed  by  means  of  chloroform. 
Hugounknq  and  Doyon2  prepared  biliverdin  from  bilirubin  by  the  action 
of  sodium  peroxide  and  a  little  acid. 

Bilifuscin,  so  named  by  Stadeler,3  is  an  amorphous  brown  pigment  soluble 
in  alcohol  and  alkalies,  nearly  insoluble  in  water  and  ether,  and  soluble  with 
great  difficulty  in  chloroform  (when  bilirubin  is  not  present  at  the  same  time). 
Pure  bilifuscin  does  not  give  Gmelin's  reaction.  This  is  also  true  for  the  bili- 
fuscin prepared  by  v.  Zumbusch,4  which  is  more  like  a  humin  substance  and 
whose  formula  is  C64H60X7O14.  Bilifuscin  has  been  found  in  gall-stones.  Bili- 
prasin  is  a  green  pigment  prepared  by  Stadeler  from  gall-stones,  which  perhaps 
is  only  a  mixture  of  biliverdin  and  bilirubin.  Dastre  and  Floresco,5  on  the 
contrary,  consider  biliprasin  as  an  intermediate  step  between  bilirubin  and  bili- 
verdin. According  to  them  it  occurs  as  a  physiological  pigment  in  the  bladder 
bile  of  several  animals  and  is  derived  from  bilirubin  by  oxidation.  This  oxidation 
is  brought  about  by  an  oxidative  ferment  existing  in  the  bile.  Bilihumin  is  the 
name  given  by  Stadeler  to  that  brownish  amorphous  residue  which  is  left  after 
extracting  gall-stones  with  chloroform,  alcohol,  and  ether.  It  does  not  give 
Gmelin's  test.  Bilicyanin  is  also  found  in  human  gall-stones  (Heinsius  and 
Campbell).  Cholohotmatin,  so-called  by  MacMunn,  is  a  pigment  often  occur- 
ring in  sheep-  and  ox-bile  and  characterized  by  four  absorption-bands,  and  which 
is  formed  from  haematin  by  the  action  of  sodium  amalgam.  In  the  dried  condi- 
tion obtained  by  the  evaporation  of  the  chloroform  solution  it  is  green,  and 
in  alcoholic  solution  olive-brown.  The  crystalline  bilipurpurin  isolated  by 
Loebisch  and  Fischler  a  from  ox-bile  is  probably  related  to  this  pigment. 

Gmelin's  and  Huppert's  reactions  are  generally  used  to  detect  the 
presence  of  bile-pigments  in  animal  fluids  or  tissues.  The  first,  as  a  rule, 
can  be  performed  directly,  and  the  presence  of  proteid  does  not  interfere 
with  it,  but,  on  the  contrary,  it  brings  out  the  play  of  colors  more  strik- 
ingly. If  blood-coloring  matters  are  present  at  the  same  time,  the  bile- 
coloring  matters  are  first  precipitated  by  the  addition  of  sodium  phosphate 
and  milk  of  lime.  This  precipitate  containing  the  bile-pigments  may  be 
used  directly  in  Huppert's  reaction,  or  a  little  of  the  precipitate  may  be 
dissolved  in  Hammarsten's  reagent.  Bilirubin  is  detected  in  blood, 
according  to  Hedenius,7  by  precipitating  the  proteins  by  alcohol,  filtering 

1  Centralbl.  f.  Physiol.,  3,  222,  and  Zeitschr.  f.  physiol.  Chem.,  14. 
1  Arch,  de  Physiol.  (5),  8. 

3  Cited  from  Hoppe-Seyler,  Physiol,  u.  Path.  chem.  Analyse,  6.  Aufl.,  225. 

4  Zeitschr.  f.  physiol.  Chem.,  31. 

6  Arch,  de  Physiol.  (5),  9. 

'MacMunn,  Journ.  of  Physiol.,  6;  Loebisch  and  Fischler,  Wien.  Sitz.-Ber.,  112 
(1903). 

7  Upsala  Lakaref.  Forh.,  29,  and  Maly's  Jahresber.,  24. 


/ 


274  THE  LIVER. 

and  acidifying  the  filtrate  with  hydrochloric  or  sulphuric  acid,  and  boiling. 
The  liquid  becomes  of  a  greenish  color.  Serum  and  serous  fluids  may  be 
boiled  directly  with  a  little  acid  after  the  addition  of  alcohol. 


Besides  the  bile-acids  and  the  bile-pigments  there  occur  in  the  bile  also 
cholesterin,  lecithin,  jecorin  or  another  phosphatide,  palmitin,  stearin, 
olein,  myristic  acid  (Lassar-Cohn  x),  soaps,  ethereal  sulphuric  acids,  con- 
jugated glucuronates,  diastatic  and  proteolytic  enzymes.  Choline  and  glycero- 
phosphoric  acid,  when  they  are  present,  may  be  considered  as  decom- 
position products  of  lecithin.  Urea  occurs,  though  only  as  traces,  as  a 
physiological  constituent  of  human,  ox,  and  dog  bile.  Urea  occurs  in  the 
bile  of  the  shark  and  ray  in  such  large  quantities  that  it  forms  one  of  the 
chief  constituents  of  the  bile.2  The  mineral  constituents  of  the  bile  are, 
besides  the  alkalies,  to  which  the  bile  acids  are  united,  sodium  and  potas- 
sium chloride,  calcium  and  magnesium  phosphate,  and  iron — 0.04-0.115 
p.  m.  in  human  bile,  chiefly  combined  with  phosphoric  acid  (Young  3). 
Traces  of  copper  are  habitually  present,  and  traces  of  zinc  are  often  found. 
Sulphates  are  entirely  absent,  or  occur  only  in  very  small  amounts. 

The  quantity  of  iron  in  the  bile  varies  greatly.  According  to  Novi  it 
is  dependent  upon  the  kind  of  food,  and  in  dogs  it  is  lowest  with  a  bread 
diet  and  highest  with  a  meat  diet.  According  to  Dastre  this  is  not  the 
case.  The  quantity  of  iron  in  the  bile  varies  even  though  a  constant  diet  is 
maintained,  and  the  variation  is  dependent  upon  the  formation  and  destruc- 
tion of  blood.  According  to  Beccari  4  the  iron  does  not  disappear  from 
the  bile  in  inanition,  and  the  percentage  shows  no  constant  diminution. 
The  question  as  to  the  extent  of  elimination  by  the  bile  of  the  iron  intro- 
duced into  the  body  has  received  various  answers.  Tii£ie__isno  doubt 
that  the  liver  has__thgjproperty  of  collecting  and  retajning^ron  as  welTas 
other  metals  from  the  blood.  Certain  investigators,  such  as  Novi  and 
Kunkel,  are  of  the  opinion  that  the  iron  introduced  and  transitorily  retained 
in  the  liver  is  eliminated  by  the  bile,  while  others,  such  as  Hamburger, 
Gottlieb,  and  Anselm,5  deny  any  such  elimination  of  iron  by  the  bile. 

Quantitative  Composition  of  the  Bile.  Complete  analyses  of  human  bile 
have  been  made  by  Hoppe-Seyler  and  his  pupils.  The  bile  was  removed 
as  fresh  as  possible  from  the  gall-bladder  of  those  cadavers,  the  livers  of 
which  were  in  no  sense  pathological. 

1  Zeitschr.  f.  physiol.  Chem.,  17. 

2  Hammarsten,  ibid.,  24. 

3  Journ.  of  Anat.  and  Physiol.,  5,  158. 

*  Novi,  see  Maly's  Jahresber.,  20;  Dastre,  Arch,  de  Physiol.  (5),  3;  Beccari,  Arch. 
ital.  de  Biol.,  28. 

5  Kunkel,  Pfluger's  Arch.,  14;  Hamburger,  Zeitschr.  f.  physiol.  Chem.,  2  and  4; 
Gottlieb,  ibid.,  15;  Anselm,  "Ueber  die  Eisenausscheidung  der  Galle."  Inaug.-Diss. 
Dorpat,  1891.     See  also  the  works  cited  in  foot-note  4,  page  206. 


TAUROCHOLIC  ACID.  265 

and  then  boiled  with  water,  when  the  glycocholic  acid  dissolves  and  may 
be  obtained  from  the  filtrate  as  crystals  on  cooling.  The  glycocholeic  acid 
with  some  transformed  glycocholic  acid  (paraglycocholic  acid)  remains 
undissolved  and  may  be  purified  by  converting  it  into  the  insoluble  barium 
salt.  The  reader  is  referred  to  more  exhaustive  works  for  other  methods 
of  preparation. 

Hyo-glycocholic  Acid,  C^H^NOg,  is  the  crystalline  glycocholic  acid  obtained 
from  the  bile  of  the  pig.  It  is  very  insoluble  in  water.  The  alkali  suits,  whose 
solutions  have  an  intensely  bitter  taste,  without  any  sweetish  after-taste,  are 
precipitated  by  CaCl2,  BaCl2,  and  MgCl2,  and  may  be  salted  out  like  a  soap  by 
Na3S()4  when  added  in  sufficient  quantity.  Besides  this  acid  there  occurs  in  the 
bile  of  the  pig  still  another  glycocholic  acid  (Jolin1). 

The  glycocholate  in  the  bile  of  the  rodent  is  also  precipitated  by  the  above- 
mentioned  salts,  but  cannot,  like  the  corresponding  salt  in  human  or  ox-bile,  be 
precipitated  on  saturating  with  a  neutral  salt  (NajSOJ.  Guano  bile-acid  possibly 
belongs  to  the  glycocholic-acid  group,  and  is  found  in  Peruvian  guano,  but  has 
not  been  thoroughly  studied. 

Taurocholic  Acid.  This  acid,  which  is  found  in  the  bile  of  man,  car- 
nivora,  oxen,  and  a  few  other  herbivora,  such  as  sheep  and  goats,  has  the 
constitution  C26H45NS07.  On  boiling  with  acids  and  alkalies  it  splits  into 
cholic  acid  and  taurin. 

Taurocholic  acid  may  be  obtained,  though  only  with  difficulty,  in  fine 
needles  which  deliquesce  in  the  air  (Parke  2).  It  is  very  soluble  in  water 
and  can  hold  the  difficultly  soluble  glycocholic  acid  in  solution.  This  is 
the  reason  why  a  mixture  of  glycocholate  with  a  sufficient  quantity  of 
taurocholate,  which  often  occurs  in  ox-bile,  is  not  precipitated  by  a  dilute 
acid.  Taurocholic  acid  is  readily  soluble  in  alcohol,  but  insoluble  in  ether. 
Its  solutions  have  a  bitter-sweet  taste.  Its  salts  are,  as  a  rule,  readily  solu- 
ble in  water,  and  the  solutions  of  the  alkali  salts  are  not  precipitated  by 
copper  sulphate,  silver  nitrate,  or  sugar  of  lead.  Basic  lead  acetate  gives, 
on  the  contrary,  a  precipitate  which  is  soluble  in  boiling  alcohol. 

Taurocholic  acid  is  best  prepared  from  decolorized,  crystallized  dog-bile, 
which  contains  only  taurocholate.  The  solution  of  this  bile  is  precipitated 
by  basic  lead  acetate  and  ammonia  and  the  washed  precipitate  dissolved  in 
boiling  alcohol.  The  filtrate  is  now  treated  with  H2S,  and  this  filtrate  is 
evaporated  at  a  gentle  heat  to  a  small  volume  and  treated  with  an  excess 
of  water-free  ether.    The  acid  sometimes  partially  crystallizes. 

Cheno-taurocholic  Acid.  This  is  the  most  essential  acid  of  goose-bile  and 
has  the  formula  C29H49NSO„.  This  acid,  though  little  studied,  is  amorphous 
and  soluble  in  water  and  alcohol. 

The  taurocholic  acids  differ  from  the  glycocholic  acids  in  being  readily 
soluble  in  water.  In  the  bile  of  the  walrus,  on  the  contrary,  a  relatively 
insoluble,  readily  crystallizable  taurocholic  acid  occurs  which  can  be  pre- 

1  Zeitschr.  f.  physiol.  Chem.,  12  and  13. 

3  Hoppe-Seyler,  Med.-chem.  Untersuch.,  160. 


266  THE  LIVER. 

cipitated  from  the  solution  of  the  alkali  salts  by  the  addition  of  mineral 
acids,  similar  to  glycocholic  acid  (Hammarsten  l). 

As  repeatedly  mentioned  above,  the  two  bile-acids  split  on  boiling  with 
acids  or  alkalies  into  non-nitrogenous  cholic  acids  and  glycocoll  or  taurin. 
Of  the  various  cholic  acids  the  following  have  been  best  studied. 

Cholic  Acid  or  Cholalic  Acid.  The  ordinary  cholic  acid  obtained  as  a 
decomposition  product  of  human  and  ox-bile,  which  occurs  regularly  in  the 
contents  of  the  intestine  and  in  the  urine  in  icterus,  has,  according  to 
Strecker  and  nearly  all  recent  investigators,  the  constitution  C24H40O5= 

(  CHOH 
C20H31  <  (CH2OH)2.    According   to   Mylius,2  cholic   acid   is   a   monobasic 

(COOH 
alcohol-acid  with  one  secondary  and  two  primary  alcohol  groups.  On  oxida- 
tion it  first  yields  dehydrocholic  acid  (Hammarsten).  On  further  oxidation 
bilianic  acid,  C24H3408  (Cleve),  is  obtained,  or,  more  correctly,  according  to 
Lassar-Cohn  and  Pregl,  a  mixture  of  bilianic  and  isobilianic  acids.  On 
the  oxidation  of  bilianic  acid  it  yields  cilianic  acid  (Lassar-Cohn)  ,  whose 
formula,  according  to  Pregl,  is  C^H^Og.  On  stronger  oxidation  it  yields 
cholesterinic  acid,  which  has  not  been  carefully  studied,  and  finally  phthalic 
acid,  as  maintained  by  Senkowski,  but  not  substantiated  by  BuLNHEiMor 
Pregl.3  On  reduction  (in  putrefaction)  cholic  acid  may  yield  desoxycholic 
acid,  C24H40O4  (Mylius).  On  reduction  with  hydriodic  acid  and  red  phos- 
phorus Pregl  obtained  a  product  which  he  considers  as  a  monocarbonic  acid^ 

(CH2 
with  the  formula  C20H31  <  (CH3)2.    Senkowski  has  obtained  an  acid  with  the 

(COOH 
formula  C24H40O2,  cholylic  acid,  on  the  reduction  of  the  anhydride.4 

Cholic  acid  crystallizes  partly  in  rhombic  plates  or  prisms  with  one 
molecule  of  water  and  partly  in  larger  rhombic  tetrahedra  or  octahedra 
with  1  molecule  of  alcohol  of  crystallization  (Mylius).  These  crystals 
become  quickly  opaque  and  porcelain-white  in  the  air.  They  are  quite 
insoluble  in  water  (in  4000  parts  cold  and  750  parts  boiling),  rather  soluble 
in  alcohol,  but  soluble  with  difficulty  in  ether.  The  amorphous  cholic  acid 
is  less  insoluble.  The  solutions  have  a  bitter-sweetish  taste.  The  crys- 
tals lose  their  alcohol  of  crystallization  only  after  a  lengthy  heating  to 
100-120°  C.  The  acid  free  from  water  and  alcohol  melts  at  195°  C.  It 
forms  a  characteristic  combination  with  iodine  (Mylius). 

1  Not  published. 

2  The  important  researches  of  Strecker  on  the  bile-acids  may  be  found  in  Annal.  d. 
Chem.  u.  Pharm.,  05,  07,  and  70;   Mylius,  Ber.  d.  deutsch.  chem.  Gesellsch.,  19. 

3  Hammarsten,  Ber.  d.  deutsch.  chem.  Gesellsch.,  11;  Cleve,  Bull.  Soc.  chim.,  35; 
Lassar-Cohn,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Pregl,  Wien.  Sitz.-Ber.,  Ill,  1902;  Sen- 
kowski, Monatshefte  f.  Chem.,  17;  Bulnheim,  Zeitschr.  f.  physioL  Chem.,  25,  in  which  the 
literature  on  cholesterinic  acid  may  be  found. 

4  Mylius,  1.  c;  Pregl,  Pniiger's  Arch.,  71;  Senkowski,  Monatshefte  f.  Chem.,  19. 


BILE  AND  ITS  FORMATION.  277 

predominates,  and  in  a  few  cases  the  latter  occurs  almost  alone.  The  bile 
of  the  rabbit,  hare,  and  kangaroo  contains,  like  the  bile  of  the  pig,  almost 
exclusively  glycocholic  acid.  A  distinct  influence  on  the  relative  amounts 
of  the  two  bile-acids  under  different  diets  has  not  been  detected.  RItter  l 
claims  to  have  found  a  decrease  in  the  quantity  of  taurocholic  acid  in  calves 
when  they  pass  from  the  milk  to  the  vegetable  diet. 

In  the  above-mentioned  calculation  of  the  taurocholic  acid  from  the 
quantity  of  sulphur  in  the  bile-salts  it  must  be  remarked  that  no  exact  con- 
clusion can  be  drawn  from  such  a  determination  since  it  is  known  that 
other  kinds  of  bile  (e.g.,  human  and  shark-bile)  contain  sulphur  in  com- 
binations other  than  taurocholic  acid.2 

The  cholesterin,  which,  according  to  several  investigators,  not  only  origi- 
nates from  the  liver,  but  also  from  the  biliary  passages,  occurs  in  larger  quan- 
tities in  the  bladder-bile  than  in  the  liver-bile,  and  is  present  to  a  greater 
extent  in  the  non-filtered  than  in  the  filtered  bile  (Doyon  and  Dufourt  3). 

The  gases  of  the  bile  consist  of  a  large  quantity  of  carbon  dioxide,  which 
increases  with  the  amount  of  alkalies,  only  traces  of  oxygen,  and  a  very 
small  quantity  of  nitrogen. 

Little  is  known  in  regard  to  the  properties  of  the  bile  in  disease.  The  quantity 
of  urea  is  found  to  be  considerably  increased  in  uraemia.  Leuein  and  tyrosin  are 
observed  in  acute  yellow  atrophy  of  the  liver  and  in  typhoid.  Traces  of  albumin 
(without  regard  to  nucleoalbumin)  have  several  times  been  found  in  the  human 
bile.  The  so-called  pigmentary  acholia,  or  the  secretion  of  a  bile  containing 
bile-acids  but  no  bile-pigments,  has  also  been  repeatedly  noticed.  In  all  such 
cases  observed  by  Rittek  he  found  a  fatty  degeneration  of  the  liver-cells,  in 
return  for  which,  even  in  excessive  fatty  infiltration,  a  normal  bile  containing  pig- 
ments was  secreted.  The  secretion  of  a  bile  nearly  free  from  bile-acids  has  been 
observed  by  Hoppe-Seyler  4  in  amyloid  degeneration  of  the  liver.  In  animals, 
dogs,  and  especially  rabbits  it  has  been  observed  that  the  blood-pigments  pass 
into  the  bile  in  poisoning  and  other  cases,  causing  a  destruction  of  the  blood-cor- 
puscles, as  also  after  intravenous  haemoglobin  injection  (Wkrtheimer  and  Meyer, 
Filehne,  Stern  5).  Bauer0  has  found  by  observations  on  man  and  dogs  that 
no  sugar  occurs  in  the  bile  either  in  alimentary  glycosuria  or  phlorhizin  diabetes, 
but  it  does  occur  during  the  first  days  of  pancreatic  diabetes.  Ethyl  alcohol 
and  more  abundantly  amyl  alcohol  pass  into  the  bile  and  cause  an  irritation  upon 
the  liver  parenchyma  which  leads  to  the  elimination  of  coagulable  proteid. 

The  physiological  secretion  of  the  gall-bladder  is  according  to  Wahl- 
gren  7  in  man  a  viscous,  alkaline  fluid  with  11.24-19.63  p.  m.  solids.     The 

1  Cited  from  Maly's  Jahresber.,  6,  195. 

2  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  32. 

3  Arch,  de  Physiol.  (5),  8. 

4  Ritter,  Compt.  rend.,  74,  and  Journ.  de  1'anat.  et  de  la  physiol.  (Robin),  1872; 
Hoppe-Seyler,  Physiol.  Chem.,  317. 

5  Wertheimer  and  Meyer,  Compt.  rend.,  108;  Filehne,  Virchow's  Arch.,  121;  Stern, 
ibid.,  123. 

"Zeitschr.  f.  physiol.  Chem.,  40. 
7  See  Maly's  Jahresber.,  32. 


27S  THE  LIVER. 

mucilaginous  properties  are  not  due  to  mucin  but  to  a  phosphorized  pro- 
tein substance  (nucleoalbumin  or  nucleoproteid). 

Instead  of  bile  there  is  sometimes  found  in  the  gall-bladder  under  pathological 
conditions  a  more  or  less  viscous,  thready,  colorless  fluid  which  contains  pseudo- 
mucins  or  other  peculiar  protein  substances.1 

Chemical  Formation  of  the  Bile.  The  first  question  to  be  answered  is 
the  f  olio  wing :  Do  the  specific  constituents  of  the  bile,  the  bile-acids  and 
bile-pigments,  originate  in  the  liver;  and  if  this  is  the  case,  do  they  come 
from  this  organ  only,  or  are  they  also  formed  elsewhere? 

The  investigations  of  the  blood,  and  especially  the  comparative  investi- 
gations of  the  blood  of  the  portal  and  hepatic  veins  under  normal  conditions, 
have  not  given  any  answer  to  this  question.  To  decide  this,  therefore,  it 
is  necessarv  to  extirpate  the  liver  of  animals  or  isolate  it  from  the  circula- 
tion. If  the  bile  constituents  are  not  formed  in  the  liver,  or  at  least  not 
alone  in  this  organ,  but  only  eliminated  from  the  blood,  then,  after  the 
extirpation  or  removal  of  the  liver  from  the  circulation,  an  accumulation  of 
the  bile  constituents  is  to  be  expected  in  the  blood  and  tissues.  If  the  bile 
constituents,  on  the  contrary,  are  formed  exclusively  in  the  liver,  then  the 
above  operation  naturally  would  give  no  such  result.  If  the  ductus  chole- 
dochus  is  tied,  then  the  bile  constituents  will  be  collected  in  the  blood  or 
tissues  whether  they  are  formed  in  the  liver  or  elsewhere. 

From  these  principles  Kobneb  has  tried  to  demonstrate  by  experiments 
on  frogs  that  the  bile-acids  are  produced  exclusively  in  the  liver.  While  he 
was  unable  to  detect  any  bile-acids  in  the  blood  and  tissues  of  these  animals 
after  extirpation  of  the  liver,  still  he  was  able  to  discover  them  on  tying  the 
ductus  choledochus.  The  investigations  of  Ludw^  and  Fleischl  2  show 
that  in  the  dog  the  bile-acids  originate  in  the  liver  alone.  After  tying  the 
ductus  choledochus  they  observed  that  the  bile  constituents  were  absorbed 
by  the  lymphatic  vessels  of  the  liver  and  passed  into  the  blood  through  the 
thoracic  duct.  Bile-acids  could  be  detected  in  the  blood  after  such  an 
operation,  while  they  could  not  be  detected  in  the  normal  blood.  But 
when  the  common  bile  and  thoracic  ducts  were  both  tied  at  the  same  time, 
then  not  the  least  trace  of  bile-acids  could  be  detected  in  the  blood,  while 
if  thev  are  also  formed  in  other  organs  and  tissues  they  should  have  been 
present. 

From  older  statements  of  Cloez  and  Vulpian,  as  well  as  Virchow,  the  bile- 
acids  also  occur  in  the  suprarenal  capsule.     These  statements  have  not  been 
-med  by  later  investigations  of  Stadelmann  and  Beier.3    At  the  present 
time  there  is  no  ground  for  supposing  that  the  bile-acids  are  formed  elsewhere 
than  in  the  liver. 


•  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  21;  Sollmann,  Amer.  Medicine,  o  (1903). 
!  Kobner,  see  Heidenhain,  Physiologie    der  Absonderungsvorgiinge  in  Hermann's 
Handbuch,  5;  Fleischl,  Arbeiten  aus  der  physiol.  Anstalt  zu  Leipzig,  Jahrgang,  9. 
'  Zeitschr.  f.  physiol.  Chem.,  IS,  in  which  the  older  literature  may  be  found. 


CHEMICAL  FORMATION  OF  BILE.  279 

It  has  been  indubitably  proved  that  the  bilc-pujments  may  be  formed  in 
other  organs  besides  the  liver,  for,  as  is  generally  admitted,  the  coloring- 
matter  hsematoidin,  which  occurs  in  old  blood  extravasations,  Ls  identical 
with  the  bile-pigment,  bilirubin  (see  page  269).  Latschexberger  *  has 
also  observed  in  horses,  under  pathological  conditions,  a  formation  of  bile- 
pigments  from  the  blood-coloring  matters  in  the  tissues.  Also  the  occur- 
rence of  bile-pigmentfl  in  the  placenta  seems  to  depend  on  their  formation 
in  that  organ,  while  the  occurrence  of  small  quantities  of  bile-pigmei 
the  blood-serum  of  certain  animals  probably  depends  on  an  absorption  of 
the  same. 

Although  the  bile-pigments  may  be  formed  in  other  organs  besides  the 
liver,  still  it  Ls  of  first  importance  to  know  what  bearing  this  organ  has  on 
the  elimination  and  formation  of  bile-pigments.  In  this  regard  it  must  be 
recalled  that  the  liver  is  an  excretory  organ  for  the  bile-pigments  circulat- 
ing in  the  blood.  Tarchaxoff  has  observed,  in  a  dog  with  biliary  fistula, 
that  intravenous  injection  of  bilirubin  causes  a  very  considerable  increase  in 
the  bile-pigments  eliminated.  This  statement  has  been  confirmed  lately 
by  the  investigations  of  Vossius.2 

Numerous  experiments  have  been  made  to  decide  the  question  whether 
the  bile-pigments  are  only  eliminated  by  the  liver  or  whether  they  are  also 
formed  therein.  By  experimenting  on  pigeons  Sterx  was  able  to  detect 
bile-pigments  in  the  blood-serum  five  hours  after  tying  the  biliary  pass 
alone,  while  after  tying  all  the  vessels  of  the  liver  and  also  the  biliary  pas- 
sages no  bile-pigments  could  be  detected  either  in  the  blood  or  the  tissues 
of  the  animal,  which  was  killed  10-2-4  hours  after  the  operation.  MIN- 
KOWSKI and  Xauxyx  3  have  also  found  that  poisoning  with  arseniuretted 
hydrogen  produces  a  liberal  formation  of  bile-pigments  and  the  secretion, 
after  a  short  time,  of  a  urine  rich  in  biliverdin  in  previously  healthy  geese. 
In  geese  with  extirpated  livers  this  does  not  occur. 

No  such  experiments  can  be  carried  out  on  mammalia,  as  they  do  not 
live  long  enough  after  the  operation;  still  there  is  no  doubt  that  this  organ 
is  the  chief  seat  of  the  formation  of  bile-pigments  under  physiological  con- 
ditions. 

In  regard  to  the  materials  from  which  the  bile-acids  are  produced,  it 
may  be  said  with  certainty  that  the  two  components,  glycocoll  and  taurin, 
which  are  both  nitrogenized,  are  formed  from  the  protein  bodies.  The 
close  relationship  of  taurin  to  the  cystin  group  of  the  proteid  molecule 
has  been  especially  shown  by  the  investigations  of  Friedmaxx  (see  Chapter 
II),  and  very  recently  Bergmaxx  4  has  shown  by  feeding  dogs  with  sodium 

1  See  Maly's  Jahresber.,  16,  and  Monatshefte  f.  Chem.,  9. 

2  Tarchanoff ,  Pfliiger's  Arch.,  9;  Vossius,  cited  from  Stadelmann,  Der  Icterus. 
'Stern,  Arch.  f.  exp.  Path.  u.  Phartn.,  19;   Minkowski  and  Naunyn,  ibid. 

*  Hcfmeister's  Beitruge,  4.     See  also  Wohlgemuth,  Zeitschr.  f.  physiol.  Chem.,  40. 


2S0  THE  LIVER. 

cholate  and  cystin  that  the  animal  body  can  transform  cystin  into  taurin 
and  that  the  taurin  of  the  bile  originates  from  the  proteids  of  the  food.  In 
regard  to  the  origin  of  the  non-nitrogenized  cholic  acid,  which  was  formerly 
considered  as  originating  from  the  fats,  nothing  is  known  pos  tively. 

The  blood-coloring  matters  are  considered  as  the  mother-substances  of 
the  bile-pigments.  If  the  identity  of  haematoidin  and  bilirubin  was  settled 
beyond  a  doubt,  then  this  view  might  be  considered  as  proved.  Independ- 
ently, however,  of  this  identity,  which  is  not  admitted  by  all  investigators, 
the  view  that  the  bile-pigments  are  derived  from  the  blood-coloring  matters 
has  strong  arguments  in  its  favor.  It  has  been  shown  by  several  experi- 
menters that  a  yellow  or  yellowish-red  pigment  can  be  formed  from  the 
blood-coloring  matters,  which  gives  Gmelin's  test,  and  which,  though  it 
may  not  form  a  complete  bile-pigment,  is  at  least  a  step  in  its  formation 
(Latschenberger)  .  A  further  proof  of  the  formation  of  the  bile-pigments 
from  the  blood-coloring  matters  consists  in  the  fact  that  haematin  on  reduc- 
tion yields  urobilin,  which  is  identical  with  hydrobilirubin  (see  Chapter 
XV).  Further,  hsematoporphyrin  (see  page  180)  and  bilirubin  are  isomers, 
according  to  Nencki  and  Sieber,  and  nearly  allied.  The  formation  of 
bilirubin  from  the  blood-coloring  matters  is  shown,  according  to  the  obser- 
vations of  several  investigators,1  by  the  appearance  of  free  haemoglobin  in 
the  plasma — produced  by  the  destruction  of  the  red  corpuscles  by  widely 
differing  influences  (see  below)  or  by  the  injection  of  haemoglobin  solution, 
causing  an  increased  formation  of  bile-pigments.  The  amount  of  pig- 
ments in  the  bile  is  not  only  considerably  increased,  but  the  bile-pigments 
may  even  pass  into  the  urine  under  certain  circumstances  (icterus).  After 
the  injection  of  haemoglobin  solution  into  a  dog  either  subcutaneously  or  in 
the  peritoneal  cavity,  Stadelmann  and  Gorodecki  2  observed  in  the  secre- 
tion of  pigments  by  the  bile  an  increase  of  61  per  cent,  which  lasted  for 
more  than  twenty-four  hours. 

If,  then,  iron-free  bilirubin  is  derived  from  the  haematin  containing  iron, 
then  iron  must  be  split  off.  This  process  may  be  represented  by  the  follow- 
ing formula,  according  to  Nencki  and  Sieber:  3  C32H32N404Fe+2II20  — Fe 
=  2C16H18N203.  The  question  in  what  form  or  combination  the  iron  is 
split  off  is  of  special  interest,  and  also  whether  it  is  eliminated  by  the  bile. 
This  latter  does  not  seem  to  be  the  case,  at  least  to  any  great  extent.  In  100 
parts  of  bilirubin  which  are  eliminated  by  the  bile  there  are  only  1.4-1.5 
parts  iron,  according  to  Kunkel;  while  100  parts  haematin  contain  about  9 
parts  iron.     Minkowski  and  Baserin  4  have  also  found  that  the  abundant 

1  See  Stadelmann,  Der  Icterus,  etc.     Stuttgart,  1891. 

2  Ibid. 

8  Arch.  f.  exp.  Path.  u.  Pharm.,  24,  440. 

4  Kunkel,  Pfliiger's  Arch.,  14;  Minkowski  and  Baserin,  Arch.  f.  exp.  Path.  u. 
Pharm.,  23. 


CHEMICAL  FORMATION  OF  BILE.  281 

formation  of  bile-pigments  occurring  in  poisoning  by  arseniuretted  hydro- 
gen does  not  increase  the  quantity  of  iron  in  the  bUe.  The  quantity  appar- 
ently does  not  seem  to  correspond  with  that  in  the  decompose!  blood-color- 
ing matters.  It  follows  from  the  researches  of  several  investigators r 
that  the  iron  is",  at  least  chiefly,  retained  by  the  liver  as  a  ferruginous  pig- 
ment or  protein  substance. 

What  relationship  does  the  formation  of  bile-acids  bear  to  the  forma- 
tion of  bile-pigments?  Are  these  two  chief  constituents  of  the  bile  derived 
simultaneously  from  the  same  material,  and  can  we  detect  a  certain  connec- 
tion between  the  formation  of  bilirubin  and  bile-acids  in  the  liver?  The 
investigations  of  Stadelmann  teach  us  that  this  is  not  the  case.  "With 
increased  formation  of  bile-pigments  the  bile-acids  decrease  and  the  supply 
of  haemoglobin  to  the  liver  acts  in  strongly  increasing  the  formation  of 
bilirubin,  but  simultaneously  strongly  decreases  the  production  of  bile- 
acids.  According  to  Stadelmann  the  formation  of  bile-pigments  and 
bile-acids  is  due  to  a  special  activity  of  the  cells. 

An  absorption  of  bile  from  the  liver  and  the  passage  of  the  bile  con- 
stituents into  the  blood  and  urine  occurs  in  retarded  discharge  of  the  bile, 
and  usually  in  different  forms  of  hepatogenic  icterus.  But  bile-pigments 
may  also  pass  into  the  urine  under  other  circumstances,  especially  in 
animals  where  a  solution  or  destruction  of  the  red  blood-corpuscles  takes 
place  through  injection  of  water  or  a  solution  of  biliary  salts,  through 
poisoning  by  ether,  chloroform,  arseniuretted  hydrogen,  phosphorus,  or 
toluylcndiamine,  and  in  other  cases.  This  occurs  also  in  man  in  severe 
infectious  diseases.  One  must  also  admit  of  a  transformation  of  blood- 
pigments  into  bile-pigments  elsewhere  than  in  the  liver,  namely,  in  the 
blood.  Such  a  belief  has  been  made  very  probable  by  the  important 
researches  of  Minkowski  and  Nauntn,  Afanassiew,  Silbermann,3  and 
in  the  above-mentioned  cases,  as  after  poisoning  with  phosphorus,  tolu- 
ylcndiamine, and  arseniuretted  hydrogen  it  has  been  confirmed  by  direct 
experiment. 

The  icterus  is  also  in  these  cases  hepatogenic ;  it  depends  upon  an  absorp- 
tion of  bile-pigments  from  the  liver,  and  this  absorption  seems  to  originate 
in  the  different  cases  in  somewhat  different  ways.  Thus  the  bile  may  be 
viscous  and  cause  a  congestion  of  the  bile  by  counteracting  the  low  secretion 
pressure.  In  other  cases  the  fine  biliary  passages  may  be  compressed  by 
an  abnormal  swelling  of  the  liver-cells,  or  a  catarrh  of  the  bile-passages 
may  occur,  causing  a  congestion  of  the  bile  (Stadelmann). 

1  See  Naunyn  and  Minkowski,  Arch.  f.  exp.  Path.  u.  Phann.,  21;  Latschenberger, 
1.  c;    Neumann,  Virchow's  Arch.,  Ill,  and  the  literature  in  foot-note  4,  page  210. 

2  The  literature  belonging  to  this  subject  is  found  in  Stadelmann,  Der  Icterus,  etc. 
Stuttgart,  1891. 


282  THE  LIVER. 

Bile  Concretions. 

The  concrements  which  occur  in  the  gall-bladder  vary  considerably  in 
size,  form,  and  number,  and  are  of  three  kinds,  depending  upon  the  kind 
and  nature  of  the  bodies  forming  their  chief  mass.  One  group  of  gall- 
stones contains  lime-pigment  as  chief  constituent,  the  other  cholesterin, 
and  the  third  calcium  carbonate  and  phosphate.  The  concrements  of  the 
last-mentioned  group  occur  very  seldom  in  man.  The  so-called  cholesterin- 
stones  are  those  which  occur  most  frequently  in  man,  while  the  lime-pigment 
stones  are  not  found  very  often  in  man,  but  often  in  oxen. 

The  figment-stones  are  generally  not  large  in  man,  but  in  oxen  and 
pigs  they  are  sometimes  found  the  size  of  a  walnut  or  even  larger.  In 
most  cases  they  consist  chiefly  of  bilirubin-calcium  with  little  or  no  bili- 
verdin.  Sometimes  also  small  black  or  greenish-black,  metallic-looking 
stones  are  found,  which  consist  chiefly  of  bilifuscin  along  with  biliverdin. 
Iron  and  copper  seem  to  be  regular  constituents  of  pigment-stones.  Man- 
ganese and  zinc  have  also  been  found  in  a  few  cases.  The  pigment-stones 
are  generally  heavier  than  water. 

The  cholesterin-stones,  whose  size,  form,  color,  and  structure  may  vary 
greatly,  are  often  lighter  than  water.  The  fractured  surface  is  radiated, 
crystalline,  and  frequently  shows  crystalline,  concentric  layers.  The 
cleavage  fracture  is  waxy  in  appearance,  and  the  fractured  surface  when 
rubbed  by  the  nail  also  becomes  like  wax.  By  rubbing  against  each  other 
in  the  gall-bladder  they  often  become  faceted  or  take  other  remarkable 
shapes.  Their  surface  is  sometimes  nearly  white  and  waxlike,  but  generally 
their  color  is  variable.  They  are  sometimes  smooth,  in  other  cases  they  are 
rough  or  uneven.  The  quantity  of  cholesterin  in  the  stones  varies  from 
642-981  p.  m.  (Ritter  1).  The  cholesterin-stones  also  sometimes  contain 
variable  amounts  of  lime-pigments,  which  give  them  a  very  changeable 
appearance. 

Cholesterin,  C27H460  (Obermuller),  or,  as  ordinarily  given, _C2yH440 
(Mauthner  and  Suida).  By  the  action  of  concentrated  sulphuric  acTcT 
or  phosphoric  acid,  and  also  in  other  ways,  hydrocarbons  are  obtained, 
which  are  called  cholcsterilin,  cholesteron,  and  cholesterilene  (Zwenger, 
Walitzky,  and  others).  Mauthner  and  Suida,2  who  have  closely  studied 
these  hydrocarbons,  have  been  able  to  prepare  a  crystalline  cholcsterilin 
by  heating  cholesterin  with  anhydrous  copper  sulphate.  The  hydrocar- 
bons stand,  according  to  Weyl,3  in  close  relationship  to  the  terpene  group. 
On  oxidation  with  hot,  strong,  nitric  acid  the  cholesterin  yields  dinitro- 

1  Journ.  de  l'anat.  et  de  la  physiol.  (Robin),  1872. 

2  Obermuller,  Du  Bois-Reymond 's  Arch.,  1889,  and  Zeitschr.  f.  physiol.  Chem.,  15; 
Mauthner  and  Suida,  Wien.  Sitzungsber.,  Math.  Nat.  Klasse,  103,  Abth.  2b,  which  also 
contains  the  older  literature. 

3  Du  Bois-Reymond 's  Arch.,  1886,  182. 


CHOLESTERIN.  283 

isopropane  (Windaus).  Otherwise  on  oxidation  cholesterin  yields  partly 
indifferent  and  partly  acid  products,  which  seem  to  indicate  a  close  relation- 
ship between  cholesterin  and  cholic  acids.  Recently  Mauthner  and 
Suida  have  obtained  three  acids  having  the  formula)  C12H1608,  C13H1808, 
and  CuH20O9  as  oxidation  products.  Diels  and  Abderhaldhx  '  obtained 
a  crystalline  acid  having  the  formula  C^H^C^  by  the  action  of  sodium 
hypobromite  upon  cholesterin.  This  acid  melts  at  290°  C.  and  yields  crys- 
talline derivatives. 

Cholesterin  occurs  in  small  amounts  in  nearly  all  animal  fluids  and 
juices.  It  occurs  only  rarely  in  the  urine,  and  then  in  very  small  quanti- 
ties. It  is  also  found  in  the  different  tissues  and  organs,  especially  abun- 
dant in  the  brain  and  the  nervous  system;  further,  in  the  yoke  of  the  egg, 
in  semen,  in  wool-fat  (together  with  isocholesterin) ,  and  in  sebum.  It 
appears  also  in  the  contents  of  the  intestine,  in  excrements,  and  in  the 
meconium.  It  especially  occurs  pathologically  in  gall-stones,  as  well  as  in 
atheromatous  cysts,  in  pus,  in  tuberculous  masses,  old  transudates,  cystic 
fluids,  sputum,  and  tumors.  It  does  not  exist  free  in  all  cases;  for  exam- 
ple, it  exists  in  part  as  fatty-acid  esters  in  wool-fat,  blood,  lymph,  brain, 
vernix  caseosa,  and  epidermis  formations.  Several  kinds  of  cholesterin, 
called  phytosterines,  have  been  found  in  the  vegetable  kingdom. 

Cholesterin  which  crystallizes  from  warm  alcohol  on  cooling  and  that 
which  is  present  in  old  transudates  contains  1  molecule  of  water  of  crystal- 
lization, melts  at  145°  C,  and  forms  colorless,  transparent  plates  whose 
sides  and  angles  frequently  appear  broken  and  whose  acute  angle  is  oTterf 
li'S  'MV  or  87°  30/.  InJajse_j43iant.itie&Jt_apgears  as  a  mass  of  white  plates 
which  shine  like  mother-of-pearl  and  have  a  greasy  feeling. 

Cholesterin  is  insoluble  in  water,  dilute  acids,  and  alkalies.  It  is  neither 
dissolved  nor  changed  by  boiling  caustic  alkali.  It  is  easily  soluble  in  boil- 
ing alcohol  and  crystallizes  on  cooling.  It  dissolves  readily  in  ether, 
chloroform,  and  benzene,  and  also  in  the  volatile  or  fatty  oils.  It  is  dis- 
solved to  a  slight  extent  by  alkali  salts  of  the  bile-acids.  The  solutions  in 
ether  and  chloroform  are  lsevorotatory. 

Among  the  many  combinations  of  cholesterin  studied  by  Obermuller 
the  propionic  ester  C2H5.CO.O.C27H45  is  of  special  interest  because  of  the 
behavior  of  the  fused  combination  on  cooling,  and  it  is  used  in  the  detection 
of  cholesterin.  For  the  detection  of  cholesterin  use  is  made  of  its  reaction 
with  concentrated  sulphuric  acid,  which  gives  colored  products. 

If  a  mixture  of  five  parts  sulphuric  acid  and  one  part  water  acts  on\ 
a  cholesterin  crystal,  this  crystal  will  show  colored  rings,  first  a  bright 
carmine-red  and  then  violet.     This  fact  is  employed  in  the  microscopic  ' 

1  Windaus,  Biochem.  Centralbl.,  1,  385;  Mauthner  and  Suida,  Wien.  Sitz-Ber. 
Math.  Nat.  Klasse,  112,  Abth.  116,  1903;  Diels  and  Abderhalden,  Ber.  d.  d.  chem. 
Gesellsch.,  36. 


284  THE  LIVER. 

detection  of  cholesterin.     Another  test,  and  one  very  good  for  the  micro 

scopical  detection  of  cholesterin,  consists  in  treating  the  crystals  first  with 

L  the  above  dilute  acid  and  then  with  some  iodine  solution.    The  crystals 

^will  be  gradually  colored  violet,  bluish  green,  and  a  beautiful  blue. 

Salkowski  's  *  Reaction.  The  cholesterin  is  dissolved  in  chloroform  and 
then  treated  with  an  equal  volume  of  concentrated  sulphuric  acid.  The 
cholesterin  solution  becomes  first  bluish  red,  then  gradually  more  violet- 
red,  while  the  sulphuric  acid  appears  dark  red  with  a  greenish  fluorescence. 
If  the  chloroform  solution  is  poured  into  a  porcelain  dish  it  becomes  violet,  / 
then  green,  and  finally  yellow. 

Liebermann-Burchard  's  2  Reaction.  Dissolve  the  cholesterin  in  about 
2  c.  c.  chloroform  and  add  first  10  drops  of  acetic  anhydride  and  then  concen- 
trated sulphuric  acid  drop  by  drop.  The  color  of  the  mixture  will  first  be  a 
beautiful  red,  then  blue,  and  finally,  if  not  too  much  cholesterin  or  sulphuric 
acid  is  present,  a  permanent  green.  In  the  presence  of  very  little  cholesterin 
the  green  color  may  appear  immediately. 

Pure,  dry  cholesterin  when  fused  in  a  test-tube  over  a  low  flame  with  two  or 
three  drops  of  propionic  anhydride  yields  a  mass  which  on  cooling  is  first  violet,  then 
blue,  green,  orange,  carmine-red,  and  finally  copper-red.  It  is  best  to  re-fuse 
the  mass  on  a  glass  rod  and  then  to  observe  the  rod  on  cooling,  holding  it  against  a 
dark  background  (Obermuller) 

Schiff's  Reaction.  If  a  little  cholesterin  is  placed  in  a  porcelain  dish  with 
the  addition  of  a  few  drops  of  a  mixture  of  two  or  three  vols,  of  cone,  hydrochloric 
acid  or  sulphuric  acid  and  one  vol.  of  a  medium  solution  of  ferric  chloride  and 
carefully  evaporated  to  dryness  over  a  small  flame,  a  reddish-violet  residue  is 
first  obtained  and  then  a  bluish-violet. 

If  a  small  quantity  of  cholesterin  is  evaporated  to  dryness  with  a  drop  of 
concentrated  nitric  acid,  one  obtains  a  yellow  spot  which  becomes  deep  orange-red 
with  ammonia  or  caustic  soda  (not  a  characteristic  reaction). 

Koprosterin  is  the  name  given  by  Bondzynski  for  the  cholesterin  which  was 
isolated  by  him  from  human  faeces,  although  it  was  prepared  earlier  by  Flint3 
and  designated  as  stercorin.  It  dissolves  in  cold,  absolute  alcohol  and  very  readily 
in  ether,  chloroform,  and  benzene.  It  crystallizes  in  fine  needles  which  melt  at 
95-96°  C.  and  is  dextrorotatory,  «(D)  =  +  24.  It  gives  the  same  color  reactions 
as  cholesterin,  with  the  exception  that  it  does  not  give  a  reaction  with  propionic 
anhydride.  According  to  Bondzynski  and  Humnicke  it  is  a  dihydrocholesterin, 
with  the  formula  C7H4R0,  which  is  derived  in  the  human  intestine  by  the  reduc- 
tion of  ordinary  cholesterin.  These  investigators  have  found  another  cholesterin, 
Mppokoprosterin,  with  the  formula  C^H^O,  in  horses'  fseces. 

Isocholesterin    is    a    cholesterin,    so-called   by    Schulze,4    with    the    formula 


1  Pfluger's  Arch.,  6. 

2  C.  Liebermann,  Ber.  d.  deutsch.  chem.  Gesellsch.,  18,  1804;  H.  Burchard,  Beitrage 
zur  Kenntniss  der  Cholesterine.     Rostock,  1889. 

3  Bondzynski,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29;  Bondzynski  and  Humnicki, 
Zeitschr.  f.  physiol.  Chem.,  22;  Flint,  ibid.,  23,  and  Amer.  Journ.  Med.  Sciences,  1862 ; 
Muller,  Zeitschr.  f.  physiol.  Chem.,  29. 

*  Ber.  d.  deutsch.  chem.  Gesellsch.,  6;  Journal  f.  prakt.  Chem.,  N.  F.,  25;  and 
Zeitschr.  f.  physiol.  Chem.,  14,  522.  See  also  E.  Schulze  and  J.  Barbieri,  Journal  i 
prakt.  Chem.,  N.  F  ,  25,  159.  In  regard  to  the  formula  for  isocholesterin,  see  Darm- 
stadter  and  Lifschutz,  Ber.  d.  deutsch.  chem.  Gesellsch.,  31,  and  E.  Schulze,  ibid.,  1200. 


CHOLESTERIN.  285 

CaJH43OH,  which  occurs  in  wool-fat  and  is  therefore  found  to  a  great  extent  in 
so-called  lanolin.  It  gives  the  Liebermann-Burchard  reaction,  but  does  not 
give  Salkowski's  reaction.     It  melts  at  138-138.5°  C. 

The  so-called  cholesterin-stones  are  employed  in  the  preparation  of 
cholesterin.  The  powder  is  first  boiled  with  water  and  then  repeatedly 
boiled  with  alcohol.  The  cholesterin  which  on  cooling  separates  from  the 
warm  filtered  solution  is  boiled  with  a  solution  of  caustic  potash  in  alcohol  so 
as  to  saponify  any  fat.  After  the  evaporation  of  the  alcohol  the  cholesterin 
is  extracted  from  the  residue  with  ether,  by  which  the  soaps  are  not  dis- 
solved; filter,  evaporate  the  ether,  and  purify  the  cholesterin  by  recrys- 
tallization  from  alcohol  ether.  Th  cholesterin  may  b  extracted  wi  h  fat 
from  tissues  and  flu  ds  by  first  extracting  with  ether  and  then  proceeding 
as  suggested  by  Ritter.1  Th  essential  po  n.s  in  his  m  thod  consist  in 
saponifying  the  fat  with  sodium  aleoholate,  removing  the  alcohol  by  evap- 
orating to  dryness  with  NaCl,  and  finally  extracting  the  dried,  pulverized 
mass  with  ether.  After  evaporating  the  ether  the  residue  is  dissolved  in  as 
little  alcohol  as  possible  and  the  cholesterin  precipitated  by  the  addition  of 
water.  It  is  ordinarily  easily  detected  in  transudates  and  pathological 
formations  by  means  of  the  microscope. 

1  Zeitschr.  f.  physiol.  Chem.,  34. 


CHAPTER  IX. 
DIGESTION. 


The  purpose  of  the  digestion  is  to  separate  those  constituents  of  the 
food  which  serve  as  the  nutriment  of  the  body  from  those  which  are  useless, 
and  to  separate  each  in  such  a  form  that  it  may  be  taken  up  by  the  blood 
from  the  alimentary  canal  and  employed  for  the  various  purposes  in  the 
organism.  This  demands  not  only  mechanical  but  also  chemical  action. 
The  first  action,  which  is  essentially  dependent  upon  the  physical  properties 
of  the  food,  consists  in  a  tearing,  cutting,  crushing,  or  grinding  of  the  food, 
while  the  second  serves  chiefly  in  converting  the  nutritive  bodies  into  a 
soluble  and  easily  absorbed  form,  or  in  the  splitting  of  the  same  into  simpler 
combinations  for  use  in  the  animal  syntheses.  The  solution  of  the  nutritive 
bodies  may  take  place  in  certain  cases  by  the  aid  of  water  alone,  but  in  most 
cases  a  chemical  metamorphosis  or  cleavage  is  necessary;  this  is  effected  by 
means  of  the  acid  or  alkaline  fluids  secreted  by  the  glands.  /The  study  of 
the  processes  of  digestion  from  a  chemical  standpoint  must  therefore  begin 
with  the  digestive  fluids,  their  qualitative  and  quantitative  composition,  as 
well  as  their  action  on  the  nutriments  and  foods. 

I.  The  Salivary  Glands  and  the  Saliva. 

The  salivary  glands  are  partly  albuminous  glands  (as  the  parotid  in  man 
and  mammals  and  the  submaxillary  in  rabbits),  partly  mucous  glands  (as 
some  of  the  small  glands  in  the  buccal  cavity  and  the  sublingual  and  sub- 
maxillary glands  of  many  animals),  and  partly  mixed  glands  (as  the  sub- 
maxillary gland  in  man) .  The  alveoli  of  the  albuminous  glands  contain  cells 
which  are  rich  in  proteid,  but  contain  no  mucin.  The  alveoli  of  the  mucin- 
glands  contain  cells  rich  in  mucin  but  poor  in  proteid.  Cells  arranged  in 
different  ways,  but  rich  in  proteids,  also  occur  in  the  submaxillary  and 
sublingual  glands.  According  to  the  analyses  of  Oidtmann  x  the  salivary 
glands  of  a  dog  contain  790  p.  m.  water,  200  p.  m.  organic  and  10  p.  m. 
inorganic  solids,  f  Among  the  solids  we  find  mucin,  proteids,  nucleoproteidsA 
\  nuclein,  enzymes  and  their  zymogens,  besides  extractive  bodies,  leucin,  xan-  \ 
thine  bodies,  and  mineral  substances^ 


1  Cit.  from  Gorup-Besanez,  Lehrbuch.  d.  physiol.  Chera.,  4.  Aufl.,  732.    The  figures 
there  given  amount  to  1010  parts  instead  of  1000  parts. 

286 


THE  SALIVA.  287 

The  occurrence  of  a  mucinogen  has  not  been  proved.  On  the  complete  removal 
of  all  mucin  K.  HoLMGBBN1  found  no  mucinogen  in  the  submaxillary  gland  of  the 
ox,  but  a  mucin-like  gluconucleoproteid. 

The  saliva  is  a  mixture  of  the  secretion  of  the  above-mentioned  groupa 
of  glands;  therefore  it  is  proper  that  a  study  be  made  of  each  of  the  different 
secretions  by  itself  and  then  the  mixed  saliva. 

The  submaxillary  saliva  in  man  may  be  easily  collected  by  introducing 
a  canula  through  the  papillary  opening  into  Wharton's  duct. 

The  submaxillary  saliva  has  not  always  the  same  composition  or  proper- 
ties; this  depends  essentially,  as  shown  by  experiments  on  animals,  upon 
the  conditions  under  which  the  secretion  takes  place.  That  is  to  say,  the 
secretion  is  partly  dependent  on  the  cerebral  system,  through  the  facial 
fibres  in  the  chorda  tympani  and  partly  on  the  sympathetic  nervous  system, 
through  the  fibres  entering  the  vessels  in  the  gland.  In  consequence  of 
this  dependence  the  two  distinct  varieties  of  submaxillary  secretion  are 
distinguished  as  chorda-  and  sympathetic  saliva.  A  third  kind  of  saliva, 
the  so-called  paralytic  saliva,  is  secreted  after  poisoning  with  curara  or 
after  the  severing  of  the  glandular  nerves.  ■ 

The  difference  between  chorda-  and  sympathetic  saliva  (in  dogs)  con- 
sists chiefly  in  their  quantitative  constitution;  the  less  abundant  sym- 
pathetic saliva  is  more  viscous  and  richer  in  solids,  especially  in  mucin, 
than  the  more  abundant  chorda-saliva.  The  specific  gravity  of  the  chorda- 
saliva  of  the  dog  is  1.0039-1.0056,  and  contains  12-14  p.  m.  solids  (Eck- 
hard  2).  The  sympathetic  has  a  specific  gravity  of  1.0075-1.018,  with 
16-28  p.  m.  solids.  The  freezing-point  of  the  chorda-saliva  obtained  from 
dogs  on  electric  stimulation  varies,  according  to  Xolf,3  between  J=  —0.193° 
and  —0.396,  with  a  content  of  3.3-6.5  p.  m.  salts  and  4.1-11.5  p.  m.  organic 
substances.  The  osmotic  pressure  is  on  an  average  a  little  higher  than 
one  half  the  osmotic  pressure  of  the  blood-serum.  The  spontaneously 
secreted  submaxillary  saliva  is  ordinarily  somewhat  diluted.  The  gases  of 
the  chorda-saliva  have  been  investigated  by  Pfluger.4  He  found  0.5-O.S 
per  cent  oxygen,  0.9-1  per  cent  nitrogen,  and  64.73-85.13  per  cent  carbon 
dioxide — all  results  calculated  at  0°  C.  and  760  mm.  pressure.  The  greater 
part  of  the  carbon  dioxide  was  chemically  combined. 

The  two  kinds  of  submaxillary  secretion  just  n^med  have  not  thus 
far  been  separately  studied  in  man.  fThe  secretion  may  be  excited  by  an 
emotion,  by  mastication,  and  by  irritating  the  mucous  membrane  of  the 
mouth,  especially  with  acid-tasting  substances.  The  submaxillary  saliva  in 
man  is  ordinarily  clear,  rather  thin,  a  little  ropy,  and  froths  easily.     Its 

1  Upsala  Liikaref.  Forh.  (N.  F.),  2;  also  Maly's  Jahresber.,  27. 

2  Cited  from  Kiihne's  Lehrb.  d.  physiol.  Chem.,  7. 
8  See  Maly's  Jahresber.,  31,  494. 

4  Pfluger 's  Arch.,  1. 


288  DIGESTION. 

reaction  is  alkaline.!1  The  specific  gravity  is  1.002-1.003,  and  it  contains 
3.6-4.5  p.  m.  solids.1  As  organic  constituents  are  found  mucin,  traces  of 
proteid  and  diastatic  enzyme,  which  latter  is  absent  in  several  species  oo 
animals.  The  inorganic  bodies  are  alkali  chlorides,  sodium  and  magnesium 
phosphates,  besides  bicarbonates  of  the  alkalies  and  calcium.  Potassium 
sulphocyanide  occurs  in  this  saliva. 

The  Sublingual  Saliva.  The  secretion  of  this  saliva  is  also  influenced 
by  the  cerebral  and  the  sympathetic  nervous  system.  The  chorda-saliva, 
which  is  secreted  only  to  a  small  extent,  contains  numerous  salivary  corpus- 
cles, but  is  otherwise  transparent  and  very  ropy.  Its  reaction  is  alkaline 
-and  contains,  according  to  Heidenhain,2  27.5  p.  m.  solids  (in  dogs). 

The  quantity  and  composition  of  the  saliva  from  the  mucin  glands  as 
well  as  from  the  albuminous  glands,  as  Pawlow's  school  has  shown,  is 
greatly  dependent  upon  the  psychical  moment,  but  also  upon  the  kind  of 
substances  introduced  into  the  mouth.  Thus  the  researches  of  Wulfson  3 
upon  dogs  have  shown  that  the  mucin  glands  yield  on  taking  food  a  ropy 
-saliva  rich  in  mucin  and  on  irritating  the  buccal  mucous  membrane  with 
destructive  or  nauseating  substances  a  thin  saliva  poor  in  mucin  is  ob- 
tained. 

/ The  sublingual  secretion  in  man  is  clear,  mucilaginous,  more  alkaline  I 
j than  the  submaxillary  saliva,  and  contains  mucin,  diastatic  enzyme,  and] 
•  potassium  sulphocyanide. 

Buccal  mucus  can  only  be  obtained  pure  from  animals  by  the  method 
suggested  by  Bidder  and  Schmidt,  which  consists  in  tying  the  exit  to  all 
the  large  salivary  glands  and  cutting  off  their  secretion  from  the  mouth. 
The  quantity  of  liquid  secreted  under  these  circumstances  (in  dogs)  was  so 
very  small  that  the  investigators  named  were  able  to  collect  only  2  grams  of 
buccal  mucus  in  the  course  of  one  hour.  It  is  a  thick,  ropy,  sticky  liquid 
containing  mucin;  it  is  rich  in  form-elements,  above  all  in  fiat  epithelium- 
cells,  mucous  cells,  and  salivary  corpuscles.  The  quantity  of  solids  in  the 
buccal  mucus  of  the  dog  is,  according  to  Bidder  and  Schmidt,4  9.98  p.  m. 

Parotid  Saliva,  f  The  secretion  of  this  saliva  is  also  partly  dependent  on 
the  cerebral  nervous  system  (n.  glossopharyngeus)  and  partly  on  the 
sympathetic.  The  secretion  may  be  excited  by  emotions  and  by  irri- 
tation of  the  glandular  nerves,  either  directly  (in  animals)  or  reflexly,  by 
mechanical  or  chemical  irritation  of  the  mucous  membrane  of  the  mouth. 
Among  the  chemical  irritants  the  acids  take  first  placg/and  the  saliva  thus 
secreted   contains,  according  to  the  observations  of  Wulfson  upon  dogs 

'See  Maly,  "Chemie  der  Verdauungssiifte  und  der  Verdauung"  in  Hermann 'ss 
Handb.,  5,  part  II,  18.     This  article  contains  also  the  pertinent  literature. 
2  Studien  d.  physiol.  Instituts  zu  Breslau,  Heft  4. 
'See  Maly's  Jahresber.,  29,  361. 
4  Die  Verdauungssiifte  und  der  Stoffwechsel  (Mitau  and  Leipzig,  1852),  5. 


THE  SALIVA.  289 

n  a   v   wice  B8  much  organic  matter  as  the  saliva  secreted  after  taking  food. 

j  Mastication  has  great  influence  in  the  secretion  of  parotid  saliva,  and  thisl 
is  especially  marked  in  certain  herbivor^J 

Human  parotid  Baliva  may  be  readily  collected  by  the  introduction  of  a 
canula  into  Stensox's  duct.  This  saliva  is  thin,  less  alkaline  than  the 
submaxillary  saliva  (the  first  drops  are  sometimes  neutral  or  acid),  without 
special  odor  or  taste.  It  contains  a  little  proteid  but  no  mucin,  which  is  to 
be  expected  from  the  construction  of  the  gland.  It  also  contains  a  diastatic 
enzyme,  which,  however,  is  absent  in  many  animals.  The  quantity  of  solids 
varies  between  5  and  16  p.  m.  The  specific  gravity  is  1.003-1.012.  Potas- 
sium Bulphocyanide  seems  to  be  present,  though  it  is  not  a  constant  con- 
stituent. Kulz  '  found  a  maximum  of  1.46  per  cent  oxygen,  3.2  per  cent 
nitrogen,  and  in  all  66.7  per  cent  carbon  dioxide  in  human  parotid  saliva. 
The  quantity  of  firmly  combined  carbon  dioxide  was  62  per  cent. 

The  mixed  buccal  saliva  in  man  is  a  colorless,  faintly  opalescent,  slightly 
ropy,  easily  frothing  liquid  without  special  odor  or  taste.  It  is  made  turbid 
by  epithelium-cells,  mucous  and  salivary  corpuscles,  and  often  by  food 
residues.  Like  the  submaxillar}'  and  parotid  saliva,  on  exposure  to  the  air 
it  becomes  covered  with  an  incrustation  consisting  of  calcium  carbonate  and 
a  small  quantity  of  an  organic  substance,  or  it  gradually  becomes  cloudy. 
Its  reaction  is  generally  alkaline  to  litmus.  The  degree  of  alkalinity  varies 
considerably  not  only  in  different  individuals  but  also  in  the  same  individual 
during  different  parts  of  the  day,  so  that  it  is  difficult  to  state  the  average 
alkalinity.  According  to  Chittenden  and  Ely  it  corresponds  to  the  alka- 
linity of  a  0.8  p.  m.  Na_,C03  solution,  or  to  a  0.2  p.  m.  solution,  according  to 
Cohn.  The  reaction  may  also  be  acid,  as  found  by  Sticker  to  be  the  case 
some  time  after  a  meal,  but  this  is  not  true  at  least  for  all  individuals.  The 
specific  gravity  varies  between  1.002  and  1.008,  and  the  quantity  of  solids 
between  5  and  10  p.  m.  According  to  Cohn  2  the  J  =  —0.20°  on  an  average 
and  the  amount  of  NaCl  is  1.6  p.  m.  The  solids,  irrespective  of  the  form- 
constituents  mentioned,  consist  of  proteid,  mucin,  two  enzymes,  ptyalin  and 
glucose,  and  mineral  bodies.  It  is  also  claimed  that  urea  is  a  normal  con- 
stituent of  the  saliva.  The  mineral  bodies  are  alkali  chlorides,  bicarbonates 
of  the  alkalies  and  calcium,  phosphates,  and  traces  of  sulphates,  nitrites, 
ammonia,  and  sulphocyanides,  which  latter  average  about  0.1  p.  m.  (Munk 
and  others).  Smaller  quantities,  0.03-0.04  p.  m.,  are  found  in  the  saliva  of 
non-smokers  (Schneider  and  Kruger  3). 

1  Zeitschr.  f.  Biologie,  23. 

5  Chittenden  and  Ely,  Amer.  Chem.  Journ.,  4,  1883;  Chittenden  and  Richards, 
Amer.  Journ.  of  Physiol.,  1;  Strieker,  cited  from  Centralbl.  f.  Physiol.,  3,  237;  Cohn, 
Deutsch.  med.  Wochenschr.,  1900. 

5  Munk,  Virchow's  Arch.,  69;  Schneider,  Amer.  Journ.  of  Physiol.,  o;  Kruger, 
Zeitschr.  f.  Biologie,  37. 


290  DIGESTION. 

Sulphocyanides,  which,  although  not  constant,  occur  in  the  saliva  of 
man  and  certain  animals,  may  be  easily  detected  by  first  acidifying  the 
saliva  with  hydrochloric  acid  and  treating  with  a  very  dilute  solution  of 
ferric  chloride.  As  control,  especially  in  the  presence  of  very  small  quan- 
tities, it  is  best  to  compare  the  test  with  another  test-tube  containing  anf 
equal  amount  of  acidulated  water  and  ferric  chloride/  Other  methods  have 
been  suggested  by  Gscheidlen  and  Solera.  The  quantitative  estimation 
can  be  done  according  to  Munk's  *  method. 

I  Ptyalin,  or  salivary  diastase,  is  the  amylolytic  enzyme  of  the  saliva.! 
'  This  enzyme  is  found  in  human  saliva,2  but  not  in  that  of  all  animals  J 
,  especially  not  in  the  typical  carnivora.  It  occurs  not  only  in  adults,  but| 
I  also  in  new-born  infants/  In  opposition  to  Zweifel  's  views  Berger  *' 

claims  that  it  is  present  not  only  in  the  parotid  gland  of  children,  but  also  in 

the  mucin  gland. 

According  to  H.  Goldschmidt4  the  saliva  (parotid  saliva)  of  the  horse  does 
not  contain  ptyalin,  but  a  zymogen  of  the  same,  while  in  other  animals  and  man 
the  ptyalin  is  formed  from  the  zymogen  during  secretion.  In  horses  the  zymogen 
is  transformed  into  ptyalin  during  mastication,  and  bacteria  seem  to  give  the 
impulse  to  this  change.  During  precipitation  with  alcohol  the  zymogen  is  changed 
into  ptyalin. 

Ptyalin  has  not  been  isolated  in  a  pure  form  up  to  the  present  time.  It 
can  be  obtained  purest  by  Cohnheim  's  5  method,  which  consists  in  carrying 
the  enzyme  down  mechanically  with  a  calcium-phosphate  precipitate  and 
washing  the  precipitate  with  water,  which  dissolves  the  ptyalin,  and  from 
which  it  can  be  obtained  by  precipitating  with  alcohol.  For  the  study  or 
demonstration  of  the  action  of  ptyalin  one  employs  a  watery  or  glycerine 
extract  of  the  salivary  glands,  or  simply  the  saliva  itself. 

Ptyalin,  like  other  enzymes,  is  characterized  by  its  action.  This  con- 
sists in  converting  starch  into  dextrins  and  sugar.  The  process  going  on 
in  this  conversion  may  be  described  as  follows:  In  the  first  stages  soluble 
starch  or  amidulin  is  formed.  From  this  amidulin,  erythrodextrin  and 
sugar  are  produced  by  hydrolytic  cleavage.  The  erythrodextrin  then  splits 
into  a-achroodextrin  and  sugar.  From  this  achroodextrin  by  splittimg 
/?-achroodextrin  and  sugar  are  formed,  and  finally  this  /?-achroodextrin 
splits  into  sugar  and  f-achrojjdextrin/^Other  investigators  explain  this 
process  in  another  manner  (see  Chapter  III) ,  hence  the  exact  procedure  is  not 
completely  clear.     Still  the  results  are  positive  as  to  the  sugar  produced  in 

'Gscheidlen,  Maly's  Jahresber.,  4;  Solera,  see  ibid.,  7  and  8;  Munk,  Virchow's 
Arch.,  69. 

2  In  regard  to  the  variation  in  the  quantity  of  ptyalin  in  human  saliva  see :  Hof  bauer, 
Centralbl.  f.  Physiol.,  10,  and  Chittenden  and  Richards,  Amer.  Journ.  of  Physiol.,  1; 
Schule,  Maly's  Jahresber.,  29. 

8  Zweifel,  Untersuchungen  iiber  den  Verdauungsapparat  der  Neugeborenen  (Berlin, 
1874) ;  Berger,  see  Maly's  Jahresber.,  30,  399. 

*  Zeitschr.  f.  physiol.  Chem.,  10. 

'Virchow's  Arch.,  28. 


PTYALIN  AND  ITS  ACTION.  291 

this  process.  For  a  long  time  it  was  considered  that  dextrose  was  the  sugar 
formed  from  starch  and  glycogen,  but  Seegen  and  O.  Nasse  have  shown 
that  this  is  not  true. 

Musculus  and  v.  Mebing  have  shown  that  the  sugar  formed  by  the 
action  of  saliva,  amvlopsin,  and  diastase  upon  starch  and  glycogen  is  in 
;  reatest  pari  maltose.  This  has  been  substantiated  by  Brown  and  HERON. 
[y  E.  KtJLZ  and  J.  VoGEL  '  have  demonstrated  thai  in  the  Baccharifica- 
tion  of  starch  and  glycogen,  isomaltose,  maltose,  and  some  dextrose'  are 
formed,  the  varying  quantities  depending  upon  the  amount  of  ferment  and 
length  of  its  action.  The  formation  of  dextrose  is  claimed  by  Tebb, 
Rohmaxx,  and  Hamburger  2  to  be  only  a  product  of  the  inversion  of  the 
maltose  by  the  glucase.  The  action  of  small  quantities  of  acid  and  salts 
upon  the  activity  of  ptyalin,  purified  by  dialysis,  has  been  studied  by  Cole.3 

The  action  of  ptyalin  in  various  reactions  has  been  the  subject  of  numer- 
ous investigations.4  Naturally  the  alkaline  saliva  is  very  active,  but 
it  is  not  as  active  as  when  neutral.  It  may  be  still  more  active  under  cir- 
cumstances in  faintly  acid  reaction,  and  according  to  Chittexdex  and 
Smith  it  acts  better  when  enough  hydrochloric  acid  is  added  to  saturate 
the  proteids  present  than  when  only  simply  neutralized.  When  the  acid- 
combined  proteid  exceeds  a  certain  amount,  then  the  diastatic  action  is 
diminished  The  addition  of  alkali  to  the  saliva  decreases  its  diastatic 
action;  on  neutralizing  the  alkali  with  acid  or  carbon  dioxide  the  retarding 
or  preventive  action  of  the  alkali  is  arrested.  According  to  Schierbeck 
carbon  dioxide  has  an  accelerating  action  in  neutral  liquids,  while  Ebstein 
claims  that  it  has  as  a  rule  a  retarding  action.  Organic  as  well  as  inorganic 
acids,  when  added  in  sufficient  quantity,  may  stop  the  diastatic  action 
^gtirely  The  degree  of  acidity  necessary  in  this  case  is  not  always  the 
same  for  a  certain  acid,  but  is  dependent  upon  the  quantity  of  ferment. 
The  same  degree  of  acidity  in  the  presence  of  large  amounts  of  ferment  has 
a  weaker  action  than  in  the  presence  of  smaller  quantities.  Hydrochloric 
acid  is  of  special  physiological  interest  in  this  regard,  namely,  it  prevents 
the  formation  of  sugar  even  in  very  small  amounts  (().():;  p.  m.).     Ilydro- 

1  Seegen,  Centralbl.  f.  d.  med.  Wissensch.,  1876,  and  Pfliiger's  Arch.,  19;  Nasse, 
ibid,,  14;  Musculus  and  v.  Mering,  Zeitschr.  f.  physiol.  Chem.,  2;  Brown  and  Heron, 
Liebig'a  Annul.,  199  and  204;   Kills  and  Vogel,  Zeitschr.  f.  Biologie,  31. 

2  Tebb,  Journ.  of  Physiol.,  15;  Rohmann,  Ber.  d.  deutsch.  chem.  Gesellsch.,  27; 
Hamburger,  Pfliiger's  Arch.,  60. 

3  Journ.  of  Physiol.,  30. 

4  See  Hammarsten,  Maly's  Jahresber.,  1;  Chittenden  and  Griswold,  Amer.  Chem. 
Journ.,  3;  Langley,  Journal  of  Physiol.,  3;  Xylen,  Maly's  Jahresber.,  12,  241;  Chit- 
tenden and  Ely,  Amer.  Chem.  Journ.,  4;  Langley  and  Eves,  Journal  of  Physiol.,  4; 
Chittenden  and  Smith,  Yale  College  Studies,  1,  1885,  1 ;  Schlesinger,  Virchow's  Arch., 
12">;  Shierbeck,  Skand.  Arch.  f.  Physiol.,  3;  Ebstein  and  C.  Schulze,  Virchow's  Arch., 
134;  Kubel,  Pfliiger's  Arch.,  7G. 


; 


292  DIGESTION. 

chloric  acid  has  not  only  the  property  of  preventing  the  formation  of  sugar, 
but,  as  shown  by  Langley,  Nylen,  and  others,  may  entirely  destroy  the 
enzyme.  This  is  important  in  regard  to  the  physiological  significance  of 
[the  saliva.  That  boiled  starch  (paste)  is  quickly,  and  unboiled  starch  only 
J  slowly,  converted  into  sugar  is  also  of  interest.  Various  kinds  of  unboiled 
starch  are  converted  with  different  degrees  of  rapidity. 

We  have  several  series  of  investigations  upon  the  rapidity  with  which 
ptyalin  acts,  and  like  testing  enzyme  action  in  general,  they  have  not  made 
use  of  the  different  times  to  produce  equal  chemical  changes  as  a  measure  of 
the  rapidity,  but  have  selected  the  quantity  of  substance  changed  in  equal 
time.  Although  the  results  are  somewhat  divergent  it  is  possible  to  deduce 
the  following  from  the  results,  f  The  rapidity  increases,  at  least  under  con- 
*  ditions  otherwise  favorable,  with  the  amount  of  enzyme  and  with  an  increas- 
ing temperature  to  a  little  above  40°  C.  Foreign  substances,  such  as  metallic 
salts,1  have  different  effects.  Certain  salts  even  in  small  quantities  com- 
pletely arrest  the  action;  for  example  HgCl2  accomplishes  this  result  com- 
pletely by  the  presence  of  only  0.05  p.  m.  Other  salts,  such  as  magnesium 
sulphate,  in  small  quantities  (0.25  p.  m.)  accelerate,  and  in  larger  quantities 
(5  p.  m.)  check  the  action.  The  presence  of  peptone  has  an  accelerating 
action  on  the  sugar  formation  (Chittenden  and  Smith  and  others).  The 
accumulation  of  the  products  of  the  amylolytic  decomposition  also  checks  the 
action  of  the  saliva/  This  has  been  shown  by  special  experiments  made 
by  Sh.  Lea.2  He  made  parallel  experiments  with  digestions  in  test-tubes 
and  in  dialyzers,  and  found  on  the  removal  of  the  products  of  the  amyloly- 
tic decomposition  by  dialysis  that  the  formation  of  sugar  took  place 
sooner,  but  also  that  considerably  more  maltose  and  less  dextrin  were 
formed. 

To  show  the  action  of  saliva  or  ptyalin  on  starch  the  three  ordinary  tests 
for  dextrose  may  be  used,  namely,  Moore's  or  Trommer's  test  or  the 
bismuth  test  (see  Chapter  XV).  It  is  also  necessary,  as  a  control,  to  first 
tesl  the  starch-paste  and  the  saliva  for  the  presence  of  dextrose.  The 
steps  formed  in  the  transformation  of  starch  into  amidulin,  erythrodextrin, 
and  achroodextrin  may  be  shown  by  testing  with  iodine. 
r  Glw.ase  only  occurs  in  saliva  to  a  slight  extent.  It  converts  maltose ) 
into  dcxt rose^/ According  to  Sticker  3  saliva  also  has  the  power  of  splitting 
sulphuretted  hydrogen  from  the  sulphur  oils  of  radishes,  onions,  and  certain 
other  vegetables. 

The  quantitative  composition  of  the  mixed  saliva  must  vary  considerably, 
not  only  because  of  individual  differences,  but  also  because  under  varying 

'See  O.   Nasse,  Pfliiger's  Arch.,  11,  and  Chittenden  and   Painter,  Yale  College 
Studies,  1,  1885,  52;  Kubel,  Pfliiger's  Arch.,  76. 

2  Journ.  of  Physiol.,  11. 

3  Munch,  med.  Wochenschr.,  43. 


COMPOSITION  OF  THE  SALIVA. 


293" 


conditions  there  may  be  an  unequal  division  of  the  secretion  from  the  differ- 
ent glands.  We  give  below  a  few  analyses  of  human  saliva  as  examples  of 
its  composition.     The  results  are  in  parts  per  1000. 


i 

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x  a  M 

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v. 

55 

< 

■ 

■ 

z  - 
m  — 

<■  a 

<  < 
-  ~ 

Water 

Solids 

Mucus  and  epithelium 

Soluble  organic  substances. 

(  Ptyalin  of  early  investigators.) 

Sulphocyanides 

992 . 9 
7.1 

1.4 
3.8 

995.16 
4.84 

1.62 
1.34 

0.06 

1.82 

994.1 
5.9 

2.13 
1.42 

0.10 
2.19 

988.3 
11.7 

994.7 
5.3 

3.5^.4 

in 
filtered 
saliva. 

994.2 
5.8 

2.2 

3.27 

0.064 

to 
0.090 

1.4 
0.04 

Salts 

1.9 

1.03 

2.2 

Hammerhaciier  found  in  1000  parts  of  the  ash  from  human  saliva:  potash 
457.2,  soda  95.9,  iron  oxide  50.11,  magnesia  1.55,  sulphuric  anhydride  (S03)  63.8, 
phosphoric  anhydride  (P205)  188.48,  and  chlorine  183.52. 

^"lhe  quantity  of  saliva  secreted  during  twenty-four  hours  cannot  be  ex- 1 
actly  determined,  but  has  been  calculated  by  Bidder  and  Schmidt  to  be{ 
1400-1500  gramgy^The  most  abundant  secretion  occurs  during  meal-times.. 
According  to  the  calculations  and  determinations  of  Tuczek  2  in  man,  1 
gram  of  gland  yields  13  grams  of  secretion  in  the  course  of  one  hour  during 
mastication.  These  figures  correspond  fairly  well  with  those  representing 
the  average  secretion  from  1  gram  of  gland  in  animals,  namely,  14.2  grams 
in  the  horse  and  8  grams  in  oxen.  The  quantity  of  secretion  per  hour 
may  be  8  to  14  times  greater  than  the  entire  mass  of  glands,  and  there  is 
probably  no  gland  in  the  entire  body,  as  far  as  is  known  at  present — the 
kidneys  not  excepted — whose  ability  of  secretion  under  physiological  con- 
ditions equals  that  of  the  salivary  glands.  AA  remarkably  abundant  secre-/ 
tion  of  saliva  is  induced  by  pilocarpine,  while  atropine,  on  the  contrary,! 
prevents  it^^* 

That  the  secretion  of  saliva,  even  if  we  do  not  consider  such  substances 
as  ptyalin,  mucin,  and  the  like,  is  not  a  process  of  filtration^  follows  from 
many  reasons,  especially  the  following:  The  salivary  glands  have,  more- 
over, a  specific  property  of  eliminating  certain  substances,  such  as  potassium 
salts  (SALKOWSKI  3),  iodine,  and  bromine  combinations,  but  not  others, 

1  Zeitschr.  f.  physiol.  Chem.,  5.     The  other  analyses  are  cited  from  Maly,  Chemi© 
der  Verdauungssafte,  Hermann's  Handbuch  d.  Physiol.,  5,  part  II,  14. 
1  Bidder  and  Schmidt,  1.  c,  13;  Tuczek,  Zeitschr.  f.  Biologie,  12. 
8  Virchow's  Arch.,  53. 


294  DIGESTION. 

such  as  iron  combinations  and  dextrose.  It  is  also  noticeable  that  the 
saliva  is  richer  in  solids  when  it  is  eliminated  quickly  by  gradually  increased 
stimulation,  and  in  larger  quantities  than  when  the  secretion  is  slower  and 
less  abundant  (Heidenhain).  The  amount  of  salts  increases  also  to  a 
certain  degree  by  an  increasing  rapidity  of  elimination  (Heidenhain, 
Werther,  Laxgley  and  Fletcher,  Novi1). 

Like  the  secretion  processes  in  general,  the  secretion  of  saliva  is  closely 
connected  with  the  processes  in  the  cells.  The  chemical  processes  going  on 
in  these  cells  during  secretion  are  still  unknown. 

The  Physiological  Importance  of  the  Saliva.  The  quantity  of  water  in 
the  saliva  renders  possible  the  action  of  certain  bodies  on  the  organs  of 
taste,  and  it  also  serves  as  a  solvent  for  a  part  of  the  nutritive  substances. 
The  importance  of  the  saliva  in  mastication  is  especially  marked  in  her- 
bivora,  and  there  is  no  question  as  to  its  importance  in  facilitating  the  act 
of  swallowing.  The  saliva  containing  mucin  is  especially  important  in 
this  regard,  and  Pawlow's  school  has  shown  that  the  secretion  also 
regulates  itself  in  this  regard.  In  dogs  dried  bread  produced  an 
abundant  flow  of  saliva  rich  in  mucin,  while  fresh  meat  which  excited 
the  appetite,  produced  a  comparatively  smaller  secretion.  The  power 
of  converting  starch  into  sugar  is  not  inherent  in  the  saliva  of  all 
animals,  and  even  when  it  possesses  this  property  the  intensity  varies  in 
different  animals.  In  man,  whose  saliva  forms  sugar  rapidly,  a  production 
of  sur:ar  from  (boiled)  starch  undoubtedly  takes  place  in  the  mouth,  but 
how  far  this  action  proceeds  after  the  morsel  has  entered  the  stomach  de- 
pends upon  the  rapidity  with  which  the  acid  gastric  juice  mixes  with  the 
swallowed  food,  and  also  upon  the  relative  amounts  of  the  gastric  juice  and 
food  in  the  stomach.  The  large  quantity  of  water  which  is  swallowed  with 
the  saliva  must  be  absorbed  and  pass  into  the  blood,  and  it  must  go 
through  an  intermediate  circulation  in  the  organism.  Thus  the  organism 
possesses  in  the  saliva  an  active  medium  by  which  a  constant  stream,  con- 
veying the  dissolved  and  finely  divided  bodies,  passes  into  the  blood  from 
the  intestinal  canal  during  digestion. 

Salivary  Concrements.  The  so-called  tartar  is  yellow,  gray,  yellowish-gray, 
brown  or  black,  and  has  a  stratified  structure.  It  may  contain  more  than  200  p.  m. 
organic  substances,  which  consist  of  mucin,  epithelium,  and  leptothrix-chains. 
The  chief  part  of  the  inorganic  constituents  consists  of  calcium  carbonate  and 
phosphate.  The  salivary  calculi  may  vary  in  size  from  that  of  a  small  grain  to 
that  of  a  pea  or  still  larger  (a  salivary  calculus  has  been  found  weighing  18.6  grams), 
and  it  contains  a  variable  quantity  of  organic  substances  (50-380  p.  m.),  which 
remains  on  extracting  the  calculus  with  hydrochloric  acid.  The  chief  inorganic 
constituent  is  calcium  carbonate. 

1  Heidenhain,  Pfliiger's  Arch.,  17;  Werther,  ibid.,  38;  Langley  and  Fletcher,  Proc- 
Roy.  Soc,  45,  and  especially  Phil.  Trans.  Roy.  Soc.  London,  180;  Novi,  Du  Bois- 
Reymond's  Arch.,  1888. 


GLANDS  OF   THE  STOMACH  AND  GASTRIC  JUICE.  295 

II.    The  Glands  of  the  Mucous  Membrane  of  the  Stomach,  and  the 

Gastric  Juice. 

Since  of  old,  the  glands  of  the  mucous  coat  of  the  stomach  have  been 
divided  into  two  distinct  kinds.  Those  which  occur  in  the  greatest  alum- 
dance  and  win  h  have  the  greatest  size  in  the  fundus  arc  called  fundus  glands, 
also  rennin  or  pepsin  glands,  and  the  others  which  occur  only  in  the  neigh- 
In  irl iood  of  the  pylorus  have  received  the  name  of  'pyloric  glands,  sometimes 
also,  though  incorrectly,  called  mucous  glands.  The  mucous  coating  of  the 
stomach  is  covered  throughout  with  a  layer  of  columnar  epithelium,  which 
is  generally  considered  as  consisting  of  goblet  cells  that  produce  mucus  by  a 
metamorphosis  of  the  protoplasm. 

The  fundus  glands  contain  two  kinds  of  cells:  adelomorphic  or  chief 
cells,  and  delomorphic  or  parietal  cells,  the  latter  formerly  called 
rennin  or  pepsin  cells.  Both  kinds  consist  of  protoplasm  rich  in  proteids; 
but  their  relationship  to  coloring-matters  seems  to  show  that  the  protein  sub- 
stances of  both  are  not  identical.  The  nucleus  must  consist  chiefly  of 
nuclein.  Besides  the  above-mentioned  constituents  the  fundus  glands 
contain  as  more  specific  constituents  several  enzymes  or  their  zymogens 
besides  a  little  fat  and  cholesterin. 

The  pyloric  glands  contain  cells  which  are  generally  considered  as 
related  to  the  above-mentioned  chief  cells  of  the  fundus  glands.  As  these 
glands  were  formerly  thought  to  contain  a  larger  quantity  of  mucin,  they 
were  also  called  mucous  glands.  According  to  Heidenhain,  independent 
of  the  columnar  epithelium  of  the  excretory  ducts  they  take  no  part  worthy 
of  mention  in  the  formation  of  mucus,  which  according  to  his  views  is 
effected  by  the  epithelium  covering  the  mucous  membrane.  The  pyloric 
glands  also  seem  to  contain  the  zymogens  referred  to  above.  Alkali  chlo- 
.,  rides,  alkali  phosphates,  and  calcium  phosphates  are  found  in  the  mucous 
coating  of  the  stomach. 

LiF.nnRMANN  l  has  obtained  an  acid-reacting  residue  on  digesting  the  mucosa 
of  the  stomach  with  pepsin-hydrochloric  acid,  which  strangely  contained  no 
nuclein,  but  only  a  proteid  containing  lecithin,  called  leeithalbumin.  To  this 
lccithalbumin  he  ascribes  a  great  importance  in  the  secretion  of  hydrochloric 
acid. 

The  Gastric  Juice.  The  observations  of  Helm  and  Beaumont  on 
persons  with  gastric  fistula  led  to  the  suggestion  that  gastric  fistulas  be 
made  on  animals,  and  this  operation  was  first  performed  by  Bassow  :  in 
1842  on  a  dog.     Verneuil  performed  the  same  on  a  man  in  1876  with 

1  Pfliiger's  Arch.,  50. 

2  Helm,  Zwei  Krankengeschichten.  Wien,  1S03.  Cit.  from  Hermann's  Handbuch, 
5,  part  II,  39.  Beaumont,  "The  Physiology  of  Digestion,"  1833;  Bassow,  Bull,  de 
la  soc.  des  natur.  de  Moscou,  1G.  Cit.  from  Maly  in  Hermann's  Handbuch.  5,  38; 
Verneuil,  see  Ch.  Richet,  "Du  Sac  gastrique  chez  l'homme,"  etc.  (Paris,  1878),  158. 


296  DIGESTIOx, . 

successful  results.  Pawlow  *  has  recently  improved  the  surgery  of  gastric 
fistula  and  has  added  much  to  the  study  of  gastrijc  secretion. 

The  secretion  of  gastric  juice  is  not  continuous,  at  least  in  man  and  in  the 
mammals  experimented  upon.  It  only  occurs  under  psychic  influence,  and 
also  hy  stimulation  of  the  mucous  membrane.  According  to  the  ordinary  view 
this  stimulation  may  be  of  a  mechanical,  thermic,  or  chemical  nature.  Among 
the  latter  is  included  alcohol  and  ether,  which  when  in  too  great  concentra- 
tion do  not  produce  a  physiological  secretion,  but  a  transudate  of  a  neutral 
or  faintly  alkaline  fluid.  To  this  class  also  belong  certain  acids,  carbon 
dioxide,  neutral  salts,  meat  extracts,  spices,  and  other  bodies,  but  unfor- 
tunately the  reported  observations  are  uncertain  and  contradictory ;  but 
there  does  not  seem  to  be  any  doubt  that  in  man  alcohol  and  meat 
extracts  at  least  have  an  accelerating  action  upon  the  flow  of  juice. 

The  most  exhaustive  researches  on  the  secretion  of  gastric  juice  (in 
dogs) have  been  done  by  Pawlow  and  his  pupils. 

In  order  to  obtain  gastric  juice  free  from  saliva  and  food  residues  they  arranged 
besides  a  gastric  fistula  also  an  oesophageal  fistula  from  which  the  swallowed  food 
could  be  withdrawn  with  the  saliva  without  entering  the  stomach,  and  in  this 
manner  an  apparent  feeding  was  possible.  In  this  way  it  was  possible  to  study  the 
influence  of  psychical  moments  on  one  side  and  the  direct  action  of  food  on  the 
mucous  membrane  on  the  other.  After  a  method  suggested  by  Heidenhain  and 
later  improved  by  Pawlow  and  Khigine,  they  have  succeeded  in  preparing  a  blind 
sac  by  partial  dissection  of  the  fundus  part  of  the  stomach,  and  the  secretion 
processes  could  be  studied  in  this  sac  while  the  digestion  in  the  other  parts  of  the 
stomach  was  going  on.  In  this  way  they  were  able  to  study  the  action  of  different 
foods  on  the  secretion. 

The  most  essential  results  of  the  investigations  of  Pawlow  and  his 
pupils  are  as  follows :  Mechanical  stimulation  of  the  mucosa  does  not  produce 
any  secretion.  Chemical  and  mechanical  irritations  of  the  mucous  mem- 
brane of  the  mouth  cause  no  reflex  excitation  of  the  secretory  nerves  of  the 
stomach.  There  are  only  two  moments  which  cause  a  secretion,  namely, 
the  psychical  moment — the  passionate  desire  for  food  and  the  sensation  of 
satisfaction  and  pleasure  on  partaking  it — and  the  chemical  moment,  the 
action  of  certain  chemical  substances  on  the  mucous  membrane  of  the 
stomach.  The  first  moment  is  the  most  important.  The  secretion  occur- 
ring  under  its  influence  by  the  vagus  fibres  appears  earlier  than  that  pro- 
duced by  chemical  irritants,  but  always  after  a  pause  of  at  least  A.\  minutes. 
This  secretion  is  more  abundant  but  less  continuous  than  the  ' '  chemical. ' ' 
It  yields  a  more  acid  and  active  juice  than  the  latter.  As  chemical  excitants 
which  cause  a  secretion  reflexively  through  the  stomach  mucosa  we  include 
only  water  and  certain  unknown  extractive  substances  contained  in  meat 

1  Pawlow,  Die  Arbeit  der  Verdauungsdriisen  (Wiesbaden,  1898),  where  the  works 
of  his  pupils  are  also  mentioned.     See  also  Ergebnisse  der  Physiol ogie,  1.  Abt.  I. 


SECRETION  OF  GASTRIC  JUICE.  297 

and  meat  extracts,  in  impure  peptone,  and  also,  it  seems,  in  milk.  Accord- 
ing o  Herzex  and  Radzikowski  l  alcohol  is  also  a  st  ong  agent  in  pro- 
ducing a  flow  of  juice.  Carbonated  alkalies  have  a  preventive  instead  of  an 
accelerating  action  on  secretion.  Fats  have  a  retarding  action  on  the 
appearance  of  secretion  and  diminish  the  quantity  of  juice  secreted  as 
well  as  the  amount  of  enzyme.  The  substances,  such  as  egg-albumin, 
which  act  as  chemical  stimulants  cannot  be  digested  by  the  "psychical" 
secretion,  but  may  perhaps  cause  a  chemical  secretion  by  their  decomposi- 
tion products. 

The  quantity  of  juice  secreted  during  digestion  is  proportional  to  the 
quantity  of  food,  and  the  secretion  of  gastric  juice  may  also  be  influenced 
by  the  kind  of  food.  This  action  of  various  foods — meat,  bread,  and  milk — 
may  be  arranged  in  progressive  series  as  follows : 


Acidity. 

Digestive  Activity 

Duration  of  Secretion. 

1. 

Meat. 

Bread. 

Bread. 

2. 

Milk. 

Meat. 

Meat. 

3. 

Bread. 

Milk. 

Milk. 

The  acidity  is  greatest  with  a  meat  diet  and  lowest  with  bread;  the 
quantity  of  enzyme  is,  on  the  contrary,  highest  with  a  bread  diet  and 
lowest  with  milk. 

The  secretion  in  the  stomach  may  also  be  influenced  by  the  small  intes- 
tine, and  in  this  way,  as  shown  by  the  recent  investigations  of  Pawlow 
and  Wirschubski,2  the  fats  have  a  retarding  action  upon  gastric  diges- 
tion, by  acting  reflexly  upon  the  duodenal  mucosa.  According  to  Frouin 
the  food  in  the  intestine  produces  a  secretion  of  gastric  juice  which 
continues  after  the  action  of  the  psychic  moment  has  ceased.  Leconte  3 
arrived  at  similar  results,  and  he  ascribes  less  importance  to  the  chemical 
secretion  as  compared  to  the  psychic  secretion  than  Pawlow  does. 

We  know  very  little  in  regard  to  the  secretion  in  man  and  the  reports  at 
hand  are  very  contradictoiy.  An  action  of  the  psychic  moment  has  thus 
far,  in  most  cases,  not  been  confirmed  to  any  mentionable  degree.  Horn- 
rorg,  who  recently  studied  a  case  of  gastric  fistula  with  oesophageal  stricture 
in  a  boy,  could  not  observe  any  influence  of  the  psychic  excitement.  The 
chewing  of  indifferent  or  badly  tasting  bodies  had  no  action,  while  on  the 
contrary  the  chewing  of  bodies  with  a  pleasant  taste  produced  a  more  or 
lass  abundant  secretion.  That  the  preparation  of  the  food  in  the  mouth 
has  an  essential  influence  upon  the  secretion  is  proven  without  doubt,  but 
we  arc  not  united  as  to  how  this  action  takes  place.     Certain  experimenters 


1  Pfliiger's  Arch.,84,  513. 

2  Cited  from  O.  Cohnheim,  Munch,  med.  Wochenschr. ,  1902. 

3  Frouiix,  Compt.  rend.  soc.  biol.,  53;  Leconte,  La  Cellule,  17. 


298  DIGESTION. 

consider  the  secreted  and  swallowed  saliva  as  the  most  essential  factor  in 
this  action,  while  others  believe  the  chemical  action  and  the  sense  of  taste 
to  be  most  important.  Among  the  chemical  excitants  Verhaegen  !  claims 
the  extractive  bodies  of  meat  are  the  most  active. 

The  Qualitative  and  Quantitative  Composition  of  the  Gastric  Juice.  The 
gastric  juice,  which  can  hardly  be  obtained  pure  and  free  from  residues  of 
the  fcod  or  from  mucus  and  saliva,  is  a  clear,  or  only  very  faintly  cloudy, 
and  in  man  nearly  colorless  fluid  of  an  insipid,  acid  taste  and  strong  acid 
reaction.  It  contains,  as  form-elements,  glandular  cells  or  their  nuclei , 
mucus-corpuscles,  and  more  or  less  changed  columnar  epithelium. 

The  acid  reaction  of  the  gastric  juice  depends  on  the  presence  of  free 
acid,  which,  as  has  been  learned  from  the  investigations  of  C.  Schmidt, 
Richet,  and  others,  consists,  when  the  gastric  juice  is  pure  and  free  from 
particles  of  food,  chiefly  or  in  large  part  of  hydrochloric  acid.  Contejean  2 
has  regularly  found  traces  of  lactic  acid  in  the  pure  gastric  juice  of  fasting 
dogs.  After  partaking  of  food,  especially  after  a  meal  rich  in  carbohydrates, 
lactic  acid  occurs  abundantly,  and  sometimes  acetic  and  butyric  acids.  In 
new-born  dogs  the  acid  of  the  stomach.is  lactic  acid,  according  to  Gmelin.3 
The  quantity  of  free  hydrochloric  acid  in  the  gastric  juice  is,  according  to 
Pawlow  and  his  pupils,  in  dogs  5-6  p.  m.,  and  in  cats  an  average  of  5.20 
p.  m.  HC1.  In  man  the  acidity  has  been  found  to  vary  considerably,  but 
it  is  generally  calculated  as  2-3  p.  m.  HC1.  According  to  Verhaegen  's 
researches  there  is  no  doubt  that  pure  human  gastric  juice  from  perfectly 
healthy  persons  has  a  higher  acidity.  There  is  hardly  any  doubt  that  at 
least  a  part  of  the  hydrochloric  acid  of  the  gastric  juice  does  not  exist  free 
in  the  ordinary  sense,  but  combined  with  organic  substances.1 

As  chief  organic  constituent,  perfectly  fresh  gastric  juice  (of  dogs)  con- 
tains a  very  complex  substance  (or  perhaps  a  mixture  of  substances)  which 
coagulates  on  boiling  and  which  separates  on  strongly  cooling  the  juice. 
This  substance  is  considered  by  certain  experimenters  (Nencki  and  Sieber, 
and  Pawlow)  as  the  conveyor  of  the  several  ferment  actions  of  the  gastric 
juice,  i.e.,  the  pepsin  as  well  as  the  rennin  action.  Gastric  juice  also  con- 
tains lecithin  and  chlorine,  and  yields  nucleoproteid,  proteose,  nuclein  bases, 
and  pentose  as  cleavage  products  (Nencki  and  Sieber  5). 


1  Hornborg,  Bidrag  till  kannedom  om  magsaftafsondringen  hos  manniskan,  Inaug.- 
Dissert.  Helsingfors,  1903.  Different  results  have  been  recently  obtained  by  Bula- 
winzew,  Biochem.  Centralbl.,  1,  593;  Verhaegen,  "La  Cellule,"  1896  and  1897. 

2  Bidder  and  Schmidt,  Die  Verdauungssafte,  etc.,  44;  Richet,  1.  c. ;  Contejean,  Con- 
tributions a  l'6tude  de  la  physiol.  de  l'estomac.  Theses.     Paris,  1892. 

3  Pfluger's  Arch.,  90. 

4  See  Richet,  1.  c. ;  Contejean,  1.  c. ;  Verhaegen,  1.  c. ;  and  the  literature  on  the  esti- 
mation of  hydrochloric  acid  in  the  gastric  contents  (page  315). 

s  Zeitschr.  f.  physiol.  Chem.,  32. 


PEPSIN.  299 

The  specific  gravity  of  gastric  juice  is  low,  1.001-1.010.  It  is  corre- 
spondingly poor  in  solids.  ( )lder  analyses  of  gastric  juice  from  man,  the  dog, 
and  the  sheep  have  been  made  by  C.  Schmidt.1  As  these  analyses  refer  only 
to  impure  gastric  juice  they  are  of  little  value.  The  epiantity  of  solids  in 
saliva-free  gastric  juice  from  a  dog  was  27  p.  m.,  with  17.1  p.  m.  organic 
substance.  The  quantity  of  free  hydrochloric  acid  was  3.1  p.  m.  Besides 
these  Schmidt  found  NaCl  1.46;  CaCl2  0.6;  KC1  1.1;  NH4C1  0.5;  earthy 
phosphates  1.9;  and  FeP04  0.1  p.m.  Nexcki  2  found  5  milligrams  sul- 
phocyanic  acid  per  liter  of  gastric  juice  of  a  dog.  The  pure  gastric  juice 
of  another  dog  contained,  according  to  Nencki  and  Sieber,3  an  average 
of  3.06  p.  m.  solids. 

Besides  the  free  hydrochloric  acid,  pepsin,  rennin,  and  a  lipase  are  the 
other  physiologically  important  constituents  of  gastric  juice. 

Pepsin.  This  enzyme  is  found,  with  the  exception  of  certain  fishes,  in 
all  vertebrates  thus  far  investigated. 

Pepsin  occurs  in  adults  and  in  new-born  infants.  This  condition  is 
different  in  new-born  animals.  While  in  a  few  herbivora,  such  as  the 
rabbit,  pepsin  occurs  in  the  mucous  coat  before  birth,  this  enzyme  is  entirely 
absent  at  the  birth  of  those  carnivora  which  have  thus  far  been  examined, 
such  as  the  dog  and  cat. 

In  various  invertebrates  enzymes  have  also  been  found  which  have  a 
proteolytic  action  in  acid  solutions.  It  has  been  shown  that  these  enzymes, 
nevertheless,  are  not  in  all  animals  identical  with  ordinary  pepsin.  Accord- 
ing to  Klug  and  Wr6blewski  4  the  pepsins  found  in  man  and  various 
higher  animal-  are  somewhat  different.  Enzymes  also  occur  in  various 
plants  and  animal  organs;  although  not  identical  with  pepsin,  they  act  in 
acid  reaction  and  stand  to  a  certain  extent  between  pepsin  and  trypsin.  To 
this  group  belongs  Glaessxer's  pseudopepsin,  which  according  to  him  is  the 
only  peptic  enzyme  in  the  pylorus  end,  but  whose  existence  is  disputed  by 
Klug.5  The  pseudopepsin  acts,  according  to  Glaessner,  also  in  neutral 
and  alkaline  reaction  and  yields  tryptophan  among  other  cleavage  prod- 
ucts. Among  the  enzymes  of  the  mucosa  of  the  stomach  belongs  the  so- 
called  antipepsin  first  discovered  by  Daxilewsky  and  Hexsel  and  then  also 
by  Wetnland,'  and  which  has  a  retarding  action  upon  pepsin  digestion 
and.  as  is  admitted,  prevents  the  self-digestion  of  the  mucous  membrane. 

Pepsin  is  as  difficult  to  isolate  in  a  pure  condition  as  other  enzymes. 
The  pepsin  prepared  by  Brucke  and  Suxdberg  gave  negative  results  with 

'L.  c. 

1  Ber.  d.  d.  chem.  Gesellsch.,  28. 

s  Zeitschr.  f.  physiol.  Chem.,  32. 

*  Klug,  Pfliiger's  Arch.,  60;   \Vr6blewski,  Zeitschr.  f.  physiol.  Chem.,  21. 
s  Glaessner,  Hofmeister's  Beitriige,  1;  Klug,  Pfliiger's  Arch.,  92. 

•  Hensel,  see  Biochem.  Centralbl.,  1,  404;  Weinland,  Zeitschr.  f.  Biologie,  44. 


■300  DIGESTION. 

most  reagents  for  proteids,  and  showed  nevertheless  a  powerful  action,  which 
seems  to  indicate  that  it  is  very  pure.  Schoumow-Simanowski,  Nencki  and 
Sieber  and  also  Pekelharing  have  designated  as  the  true  enzyme  the  sub- 
.stance  containing  chlorine,  which  they  obtained  by  strongly  cooling  the 
gastric  juice.  That  this  substance  is  not  an  individual,  and  hence  cannot 
T^e  pepsin,  follows  from  the  investigations  of  Pekelharing.  While  pepsin, 
according  to  Nencki  and  Sieber,  was  rich  in  phosphorus  and  contained 
jiucleoproteid,  Pekelharing 's  pepsin  was  free  from  phosphorus  and 
yielded  no  nucleoproteid. 

Friedenthal  and  Miyamota  *  have  also  shown  that  the  pepsin  is 
still  active  after  the  splitting  off  of  the  nuclein  complex  (and  also  the  pro- 
teid).  The  question  as  to  the  nature  of  pepsin  has  not  been  positively 
decided,  just  as  is  the  case  with  other  enzymes.  According  to  Biernacki  2 
pepsin  in  neutral  solutions  is  destroyed  by  heating  to  55°  C.  In  the 
presence  of  2  p.  m.  HC1  a  temperature  of  55°  C.  is  without  action;  the 
pepsin  in  acid  solution  is  destroyed  by  heating  to  65°  C.  for  five  minutes. 
On  adding  peptone  and  certain  salts  the  pepsin  may  be  heated  to  70°  C. 
without  decomposing.  In  the  dry  state  it  can,  on  the  contrary,  be  heated 
to  over  100°  C.  without  losing  its  physiological  action.  The  only  property 
which  is  characteristic  of  pepsin  is  that  it  dissolves  proteid  bodies  in  acid 
but  not  in  neutral  pr  alkaline  solutions,  with  the  formation  of  proteoses, 
peptones,  and  other  products. 

The  methods  for  the  preparation  of  relatively  pure  pepsin  depend,  as  a 
rule,  upon  it?  property  of  being  thrown  down  with  finely  divided  precipi- 
tates of  other  bodies,  such  as  calcium  phosphate  or  cholesterin.  The  rather 
complicated  methods  of  Brucke  and  Sundberg  are  based  upon  this  prop- 
erty. Pekelharing  makes  use  of  a  prolonged  dialysis  and  precipitation 
with  0.2  p.  m.  HC1. 

Very  permanent  pepsin  solutions,  from  which  the  enzyme  with  con- 
siderable proteid  can  be  precipitated  by  alcohol,  may  be  prepared  by  extrac- 
tion with  glycerine.  Solutions  having  a  strong  action  may  also  be  prepared 
by  making  an  infusion  of  the  gastric  mucosa  of  an  animal  in  acidified  water 
(2-5  p.  m.  HC1).  This  is  unnecessary,  as  we  can  obtain  pure  gastric  juice 
according  to  Pawlow's  method,  and  also  because  very  active  commercial 
preparations  of  pepsin  can  be  bought  in  the  market. 

The  Action  of  Pepsin  on  Proteids.  Pepsin  is  inactive  in  neutral  or 
alkaline  reactions,  but  in  acid  liquids  it  dissolves  coagulated  proteid 
bodies.  The  proteid  always  swells  and  becomes  transparent  before  it  dis- 
solves.    Unboiled  fibrin  swells  up  in  a  solution  containing  1  p.  m.  HC1, 

1  Brucke,  Wien.  Sitzungsber. ,  43;  Sundberg,  Zeitschr.  f.  physiol.  Chem.,  9;  Schou- 
mow-Simanowski, Arch.  f.  exp.  Path.  u.  Pharm.,  33;  Pekelharing,  Zeitschr.  f.  physiol. 
Chem.,  22  and  35;  Nencki  and  Sieber,  ibid.,  32;  Friedenthal  and  Miyamota,  Centralbl. 
f  Physiol.,  15,  785. 

2  Zeitschr.  f.  Biologie,  28. 


ACTION  OF  PEPSIN.  301 

forming  a  gelatinous  mass,  and  does  not  dissolve  at  ordinary  temperature 
within  a  couple  of  days.  Upon  the  addition  of  a  little  pepsin,  however, 
this  swollen  mass  dissolves  quickly  at  ordinary  temperatures.  Hard- 
boiled-egg  albumen,  cut  in  thin  pieces  with  sharp  edges,  is  not  perceptibly 
changed  by  dilute  acid  (2-4  p.  in.  HC1)  at  the  temperature  of  the  body  in 
the  course  of  several  hours.  But  the  simultaneous  presence  of  pepsin 
causes  the  edges  to  become  clear  and  transparent,  blunt  and  swollen,  and 
the  proteid  gradually  dissolves. 

From  what  has  been  said  above  in  regard  to  pepsin,  it  follows  that 
proteids  may  be  employed  as  a  means  of  detecting  pepsin  in  liquids.  <)x- 
fibrin  may  be  employed  as  well  as  coagulated  egg  albumen,  which  latter  is 
used  in  the  form  of  slices  with  sharp  edges.  As  the  fibrin  is  easily  digested 
at  the  normal  temperature,  while  the  pepsin  test  with  egg-albumen  requires 
the  temperature  of  the  body,  and  as  the  test  with  fibrin  is  somewhat  more 
delicate,  it  is  often  preferred  to  that  with  egg-albumen.  When  we  speak  of 
the  "pepsin  test"  without  further  explanation,  we  ordinarily  understand 
it  as  the  test  with  fibrin. 

This  test,  nevertheless,  requires  care.  The  fibrin  used  should  be  ox- fibrin 
and  not  pig-fibrin,  which  last  is  dissolved  too  readily  with  dilute  acid  alone. 
The  unboiled  ox-fibrin  may  be  dissolved  by  acid  alone  without  pepsin,  but 
this  generally  requires  more  time.  In  testing  with  unboiled  fibrin  at  normal 
temperature,  it  is  advisable  to  make  a  control  test  with  another  portion  of 
the  same  fibrin  with  acid  alone.  Since  at  the  temperature  of  the  body 
unboiled  fibrin  is  more  easily  dissolved  by  acid  alone,  it  is  best  always  to 
work  with  boiled  fibrin. 

As  pepsin  has  not  thus  far  been  prepared  in  a  positively  pure  condition, 
it  is  impossible  to  determine  the  absolute  quantity  of  pepsin  in  a  liquid.  It 
is  only  possible  to  compare  the  relative  amounts  of  pepsin  in  two  or  more 
liquids,  which  may  be  done  in  several  ways. 

The  older  method,  that  of  Brucke,  consists  in  diluting  the  two  pepsin  solu- 
tions to  be  compared  with  certain  proportions  of  1  p.  m.  hydrochloric  acid,  so 
that  when  the  amount  of  pepsin  contained  in  the  original  solution  is  equal  to  1, 
each  solution  contains  a  degree  of  dilution,  p,  corresponding  to  1,  £,  \,  J,  l  .  etc. 
A  Hock  of  fibrin  or  a  piece  of  hard-boiled  egg  is  added  to  each  test  and  the  time 
noted  when  each  test  begins  to  digest  and  when  it  ends.  The  relative  amount 
of  pepsin  is  calculated  from  the  rapidity  of  digestion  as  follows:  the  tests  . 
i,,1,;  of  one  series  was  digested  in  the  same  time  as  tests  p  =  l,  },  {  of  the  other 
series;  hence  the  first  solution  contained  four  times  as  much  pepsin.  This  method 
is  not  used  as  often  as  the  following: 

M BTT  's  Method.  Draw  up  white  of  egg  in  a  glass  tube  1-2  millimeters  in  diam- 
eter, coagulate  it  by  plunging  it  into  hot  water  at  95°  ('.  and  cut  the  ends  off 
sharply;  then  add  two  tubes  to  each  test-tube  with  a  few  cubic  centimeters  of  the 
acid-pepsin  solution;  allow  them  to  digest  at  body  temperature,  and  after  a  certain 
time,  generally  after  ten  hours,  measure  the  lineal  extent  of  the  digested  layer 
of  albumen  in  the  various  tests,  bearing  in  mind  that  the  digested  layer  at  each 
end  must  not  be  longer  than  0-7  millimeters.  The  quantity  of  pepsin  in  the 
comparative  tests  is  as  the  square  of  the  millimeters  of  the  albumen-column  dis- 
solved in  the  same  time.     Thus  if  in  one  case  2  millimeters  of  albumen  were  dis- 


302  DIGESTION. 

solved  and  in  the  other  3  millimeters,  then  the  quantity  of  pepsin  is  as  4:9- 
If  the  fluid  removed  from  the  stomach,  which  is  rich  in  bodies  having  a  disturb- 
ing influence  upon  pepsin  digestion,  is  to  be  tested,  then  the  liquid  must  be  first 
properly  diluted  with  2-4  p.  m.  hydrochloric  acid  (Nierenstein  and  Schiff1). 

Objections  have  been  raised  against  these  methods  from  several  sides,  but. 
they  can  be  recommended  for  practical  purposes  as  being  simple  and  rather 
accurate.  Huppert  and  E.  Schutz  measure  the  relative  quantities  of  pepsin 
from  the  amount  of  secondary  proteoses  formed  under  certain  conditions.  The 
proteoses  were  determined  by  the  polariscope.  J.  Schutz  determines  the  total 
proteose-nitrogen  and  Spriggs  2  finds  that  the  change  in  the  viscosity  is  a 
measure  of  the  amount  of  pepsin. 

The  rapidity  of  the  pepsin  digestion  depends  on  several  circumstances. 
Thus  different  acids  are  unequal  in  their  action;  hydrochloric  acid  shows 
in  slight  concentration,  0.8-1.8  p.  m.,  a  more  powerful  action  than  any  other 
acid,  whether  inorganic  or  organic.  In  greater  concentration  other  acids  may 
have  a  powerful  action,  and  one  can  say  that,  as  a  rule,  the  acids  having 
the  greatest  avidity  have  a  greater  action  in  slight  concentration  than  wyeak 
acids.  Still  sulphuric  acid  forms  an  exception  (Pfleiderer)  .  The  state- 
ments in  regard  to  the  action  of  various  acids  are  somewhat  contradictory.3 
The  degree  of  acidity  is  also  of  the  greatest  importance.  With  hydrochloric 
acid  the  degree  of  acidity  is  not  the  same  for  different  proteid  bodies  For 
fibrin  it  is  0.8-1  p.  m.,  for  myosin,  casein,  and  vegetable  proteids  about 
lp.m.,  for  coagulated  egg  albumen,  on  the  contrary,  about  2.5  p.  m.  The 
rapidity  "of  the  digestion  increases,  at  least  to  a  certain  point,  with  the 
quantity  of  pepsin  present,  unless  the  pepsin  added  is  contaminated  by  a 
large  quantity  of  the  products  of  digestion,  which  may  prevent  its  action. 

According  to  E.  Schutz,4  whose  statements  have  been  confirmed  by 
several  others,  the  digestion  products  produced  in  a  certain  time  are,  within 
certain  limits,  proportional  to  the  square  root  of  the  relative  amounts  of 
pepsin.  The  accumulation  of  products  of  digestion  has  a  retarding  action 
on  digestion,  although,  according  to  Chittenden  and  Amerman,5  the 
removal  of  the  digestion  products  by  means  of  dialysis  does  not  essentially 
change  the  relationship  between  the  proteoses  and  true  peptones.  Pepsin  acts 
slower  'at  low  temperatures  than  it  does  at  higher  ones.  It  is  even  active 
in  the  neighborhood  of  0°C,  but  digestion  takes  place  very  slowly  at  this 
temperature.  With  increasing  temperature  the  rapidity  of  digestion  also 
increases  until  about  40°  C,  when  the  maximum  is  reached.     According 

1  Mett,  see  Pawlow,  1.  c,  31 ;   Nierenstein  and  Schiff,  Berl.  klin.  Wochenschr. ,  40. 

2  Huppert  and  Schutz,  Pfliiger's  Arch.,  80;  J.  Schutz,  Zeitschr.  f.  physiol.  Chem., 
30;  Spriggs,  ibid.,  35. 

3  See  Wroblewski,  Zeitschr.  f.  physiol.  Chem.,  21,  and  especially  Pfleiderer,  Pfliiger's 
Arch.,  06,  which  also  gives  references  to  other  works,  and  Larin,  Biochem.  Centralbl., 
1,  484. 

4  Zeitschr.  f.  physiol.  Chem.,  9 
s  Journ.  of  Physiol.,  14. 


PEPSIN  DIGESTION.  303 

to  the  investigations  of  Flaum  *  it  is  probable  that  the  relationship  between 
proteoses  and  peptones  remains  the  same,  irrespective  of  whether  the 
digestion  takes  place  at  a  low  or  high  temperature,  as  long  as  the  digestion 
is  continued  for  a  long  enough  time.  If  the  swelling  up  of  the  proteid  is 
prevented,  as  by  the  addition  of  neutral  salts,  such  as  \a(  '1  in  sufficient 
aim  units,  or  by  the  add  ion  of  bile  to  the  acid  liquid,  digestion  can  be 
prevented  to  a  greater  or  less  extent.  Foreign  bodies  of  different  kinds 
produce  different  a  tions,  in  which  naturally  the  variable  quantities  in 
which  they  are  added,  are  of  the  greatest  importance.  Salicylic  acid  and 
carbolic  acid,  and  (spec  ally  sulphates  (Pfleiderer),  retard  digestion, 
while  arsenious  acid  promotes  it  (Chittexdkx),  and  hydrocyanic  acid  is 
relatively  indifferent.  Alcohol  in  large  quantities  (10  per  cent  and  above) 
disturbs  the  digestion,  while  small  quantities  act  indifferently.  Metallic 
salts  in  very  small  quantities  may  indeed  sometimes  accelerate  digestion, 
but  otherwise  they  tend  to  retard  it.  The  action  of  metallic  salts  in  dif- 
ferent cases  can  be  explained  in  various  ways,  but  they  often  seem  to 
form  with  proteids  insoluble  or  difficultly  soluble  combinations.  The 
alkaloids  may  also  retard  the  pepsin  digestion  (Chittexdkx  and  Allen  2). 
A  very  large  number  of  observations  have  been  made  in  regard  to  the  action 
of  foreign  substances  on  artificial  pepsin  digestion,  but  as  these  observa- 
tions have  not  given  any  direct  result  in  regard  to  the  action  of  these  same 
substances  on  natural  digestion,  as  well  as  upon  secretion  and  absorp- 
tion, we  will  not  discuss  them  here. 

The  Products  of  the  Digestion  of  Proteids  by  Means  of  Pepsin  and  Acid. 
In  the  digestion  of  nucleoproteids  or  nucleoalbumins  an  insoluble  residue 
of  nuclein  or  pseudonuclein  always  remains,  although  under  certain  circum- 
stances a  complete  solution  may  occur.  Fibrin  also  yields  an  insoluble 
residue,  which  consists,  at  least  in  great  part,  of  nuclein,  derived  from  the 
form-elements  enclosed  in  the  blood-clot.  This  residue  which  remains  after 
the  digestion  of  certain  proteids  was  called  dyspeptone  by  Meissner,  In 
the  digestion  of  proteids,  substances  similar  to  acid  albuminates,  para- 
peptone  (Meissner3),  antialbumate,  and  antialbumid  (Kuhxe),  may  also  be 
formed.  On  separating  these  bodies  the  filtered  liquid,  neutralized  at 
boiling-point,  contains  proteoses  and  peptones  in  the  old  sense,  while  the 
so-called  Kuhne  t  ue  peptone  and  the  other  cleavage  products  are  only 
obtained  after  a  longer  and  more  intense  digestion.  The  relationship 
between  the  various  proteoses  changes  very  much  in  different  cases 
and   in   the   digestion  of    the  various    proteids.      For  instance,   a   larger 

1  Zeitschr.  f.  Biologie,  28. 

1  Studies  from  the  Lab.  Physiol.  Chem.  Yale  University,  1,  76.  See  also  Chitten- 
den and  Stewart,  ibid.,  3,  60. 

3  The  works  of  Meissner  on  pepsin  digestion  are  found  in  Zeitschr.  f.  rat.  Med.,  7, 
8, 10,  12,  and  14. 


304  DIGESTION. 

quantity  of  primary  proteoses  is  obtained  from  fibrin  than  from  hard- 
boiled-egg  albumen  or  from  the  proteids  of  meat ;  and  the  different  proteids, 
according  to  the  researches  of  Klug,1  yield  on  pepsin  digestion  unequal 
quantities  of  the  various  digestive  products.  In  the  digestion  of  unboiled 
fibrin  an  intermediate  product  may  be  obtained  in  the  earlier  stages  of 
the  digestion — a  globulin  which  coagulates  at  55°  C.  (Hasebroek2).  For 
information  in  regard  to  the  different  proteoses  and  peptones  which  are 
formed  in  pepsin  digestion  the  reader  is  referred  to  previous  pages  (41-44). 

Action  of  Pepsin-Hydrochloric  Acid  on  Other  Bodies.  The  gelatine- 
forming  substance  of  the  connective  tissue,  of  the  cartilage,  and  of  the  bones, 
from  which  last  the  acid  only  dissolves  the  inorganic  substances,  is  con- 
verted into  gelatine  by  digesting  with  gastric  juice.  The  gelatine  is  further 
changed  so  that  it  loses  its  property  of  gelatinizing  and  is  converted  into 
gelatoses  and  peptone  (see  page  62).  True  mucin  (from  the  submaxil- 
lary) is  dissolved  by  the  gastric  juice,  yielding  substances  similar  to  pep- 
tone and  a  reducing  substance  similar  to  that  obtained  by  boiling  with 
a  mineral  acid.  Elastin  is  dissolved  more  slowly  and  yields  the  above- 
mentioned  substances  (page  60).  Keratin  and  the  epidermal  formations 
are  insoluble.  The  nuclein  is  dissolved  with  difficulty  and  the  cell  nuclei, 
therefore,  remain  undissolved  in  great  part  in  the  gastric  juice.  The  animal 
cell-membrane  is,  as  a  rule,  more  easily  dissolved  the  nearer  it  stands  to 
elastin,  and  it  dissolves  with  greater  difficulty  the  more  closely  it  is  related 
to  keratin.  The  membrane  of  the  plant-cell  is  not  dissolved.  Oxyhcemo- 
globin  is  changed  into  hsematin  and  proteid,  the  latter  undergoing  further 
digestion.  It  is  for  this  reason  that  blood  is  changed  into  a  dark-brown 
mass  in  the  stomach.  The  gastric  juice  does  not  act  on  fat,  but,  on  the 
contrary,  dissolves  the  cell-membrane  of  fatty  tissue,  setting  the  fat 
free.  Gastric  juice  has  no  action  on  starch  or  the  simple  varieties  of  sugar. 
The  statements  in  regard  to  the  ability  of  gastric  juice  to  invert  cane- 
sugar  are  very  contradictory.  At  least,  this  action  of  the  gastric  juice  is 
not  constant,  and  if  it  is  present  at  all  it  is  probably  due  to  the  action 
of  the  acid. 

Pepsin  alone,  as  above  stated,  has  no  action  on  proteids,  and  an  acid  of  the 
intensity  of  the  gastric  juice  can  only  very  slowly,  if  at  all,  dissolve  coagulated 
albumen  at  the  temperature  of  the  body.  Pepsin  and  acid  together  not  only  act 
more  quickly,  but  qualitatively  they  act  otherwise  than  the  acid  alone,  at  least 
upon  dissolved  proteid.  This  has  led  to  the  assumption  as  to  the  presence  of  a 
pepsin-hydrochloric  acid  whose  existence  and  action  is  only  hypothetical.  As 
pepsin  digestion,  it  seems,  yields  finally  the  same  products  as  the  hydrolytic 
cleavage  with  acids,  we  can  only  say  for  the  present  that  this  enzyme  acts  like 
other  catalysators  in  very  powerfully  accelerating  a  process  which  would  proceed 
also  without  the  catalysator. 

Rennin,  or  chymosin,  is  the  second  enzyme  of  the  gastric  juice.  It 
occurs  in  the  gastric  juice  of  man  under  physiological  conditions,  but 

1  Pfluger's  Arch.,  65.  2  Zeitschr.  f.  physiol.  Chem.,  11. 


RENNIN.  305 

may  be  absent  under  special  pathological  conditions  (ScHUlfBUBO, 
Boas,  Johnson,  Klempereb1).  It  is  habitually  found  in  toe  neu- 
tral, watery  infusion  of  the  fourth  stomach  of  the  calf  and  sheep,  espe- 
cially in  an  infusion  of  the  fundus  part.  In  other  mammals  and  in  birds 
it  is  seldom  found,  and  in  fishes  hardly  ever  in  the  oeutral  infusion.  In 
these  cases,  as  in  man  and  the  higher  animals,  a  rflnnin-forming  substance, 

a  rennin  zymogen,  occurs  which  is  converted  into  rennin  by  the  action  of 
an  acid.     Enzymes  acting  like  rennin  are  also  found  in   the  blood  and 

several  organs  of  higher  animals,  as  well  as  in  invertebrates.  Similar 
enzymes  also  occur  widely  diffused  in  the  plant  kingdom,  and  numerous 
micro-organisms  have  the  power  of  producing  rennin  enzymes. 

Rennin  is  just  as  difficult  to  prepare  in  a  pure  state  as  the  other  enzymes. 
The  purest  rennin  enzyme  thus  far  obtained  did  not  give  the  ordinary 
proteid  reactions.  On  heating  its  solution  rennin  is  more  or  less  quickly 
destroyed,  depending  upon  the  length  of  heating  and  upon  the  concentration. 
If  an  active  and  strong  infusion  of  the  gastric  mucosa  in  water  contain- 
ing 3  p.  m.  IIC1  is  heated  to  37-40°  C.  for  48  hours,  the  rennin  is  destroyed, 
while  the  pepsin  remains.  A  pepsin  solution  free  from  rennin  can  be 
obtained  in  this  way.  Rennin  is  characterized  by  its  physiological  action, 
which  consists  in  coagulating  milk  or  a  casein  solution  containing  lime, 
if  neutral  or  very  faintly  alkaline.  The  law  of  the  action  of  this  enzyme  is 
different  from  the  action  of  pepsin.  As  specially  shown  "by  Fuld  ; 2  within 
certain  limits,  the  coagulation  time,  T ',  is  equal  to  a  constant,  C,  divided  by 
the  quantity  of  rennin,  L. 

From  the  different  laws  of  pepsin  and  rennin  action  it  follows  that  the  repeated 
appearance  recently  of  the  view  of  Pawlow's  school,  that  pepsin  and  rennin  are 
the  same  bodies,  cannot  be  correct.  This  also  follows  from  the  fact  that  active 
solutions  of  pepsin  may  be  prepared  which  have  no  rennin  action.  Glaessner 
has  also  isolated  the  proenzymes  of  both  bodies  and  has  shown  their  different 
division  in  the  stomach.  According  to  Nencki  and  Sieber,  with  whom  Pekel- 
HARING8  agrees,  the  enzyme  of  the  gastric  juice  forms  a  gigantic  molecule  which 
is  able  to  perform  the  different  actions  at  the  same  time  although  each  enzyme 
action  is  connected  with  a  certain  atomic  complex.  Such  a  view  might  appear 
plausible,  but  as  the  proenzymes  of  both  enzymes,  pepsin  and  rennin,  as  well  as 
the  enzymes  themselves,  can  be  separated  from  each  other,  and  as  the  body  pre- 
cipitated from  the  gastric  juice  by  cold  and  which  forms  the  ferment  has  been 
shown  to  be  a  mixture,  this  view  does  not  seem  to  be  sufficiently  well  grounded. 

Rennin  may  be  carried  down  by  other  precipitates  like  other  enzymes 
and  thus  may  be  obtained  relatively  pure.     It  may  also  be  obtained,  con- 

1  Schumburg,  Virchow's  Arch.,  97.  A  good  review  of  the  literature  may  be  found 
in  Szydlowski,  Beitrage  BUT  Kenntniss  des  Labenzym  nach  Beobachtungen  an  Saug- 
lingen,  Jahrb.  f.  Kinderheilkunde,  X.  P.,  34.  See  also  Lurcher,  Pfluger's  Arch.,  (>9, 
which  also  contains  the  pertinent  literature.  An  excellent  review  of  the  literature  on 
rennin  and  its  action  may  he  found  in  E.  Fuld,  Ergebnisse  der  Physiol.,  1,  Abt.  I,  408. 

2  Hofmeister's  Beitrage,  8. 

8  Glaessner,  ibid.,  1;   Zeitschr.  f.  physiol.  Chcm.,  32. 


306  DIGESTION. 

taminated  with  a  great  deal  of  proteids,  by  extracting  the  mucous  coat  of 
the  stomach  with  glycerine. 

A  comparatively  pure  solution  of  rennin  may  be  obtained  in  the  follow- 
ing way.  An  infusion  of  the  mucous  coat  of  the  stomach  in  hydrochloric 
acid  is  prepared  and  then  neutralized,  after  which  it  is  repeatedly  shaken 
with  new  quantities  of  magnesium  carbonate  until  the  pepsin  is  precipi- 
tated. The  filtrate,  which  should  act  strongly  on  milk,  is  precipitated  by 
basic  lead  acetate,  the  precipitate  decomposed  with  very  dilute  sulphuric 
acid,  the  acid  liquid  filtered  and  treated  with  a  solution  of  stearin  soap. 
The  rennin  is  carried  down  by  the  fatty  acids  set  free,  and  when  these  last 
are  placed  in  water  and  removed  by  shaking  with  ether,  the  rennin  remains 
in  the  watery  solution. 

Parachymosin  is  the  name  given  by  Bang  *  to  another  rennin  enzyme 
which  occurs  in  pepsin  preparations,  but  not  in  the  calf's  stomach.  The 
enzyme,  on  the  contrary,  forms  the  true  rennin  enzyme  of  the  human  and 
pig's  stomach.  Parachymosin  is  very  much  more  resistant  towards  acids 
than  calf  rennin,  but  it  is  more  readily  destroyed  by  alkalies.  Calcium 
chloride  accelerates  the  casein  coagulation  with  parachymosin  very  much 
more  than  with  chymosin. 

Plastein.  As  mentioned  on  page  44  Danilewsky  first  showed  the 
power  of  rennin  solution  of  causing  a  partial  coagulation  of  proteoses  and  of 
converting  them  into  so-called  plastein.  This  action,  which  is  also  ascribed 
to  other  enzyme  'solutions  (see  page  44)  has  probably  nothing  to  do  with 
the  rennin  enzyme,  but  depends  more  likely  upon  another  enzyme.  The 
nature  of  these  enzymes,  as  well  as  the  manner  and  importance  of  the 
plastein  formation,  is  still  unknown. 

Gastric  Lipase  (stomach  steapsin).  F.  Volhard  2  has  made  the  dis- 
covery that  the  gastric  juice  had  a  strong  fat-splitting  action  only  when  the 
fat  was  in  a  fine  emulsion,  as  in  the  yolk  of  the  egg,  milk,  or  cream.  This 
action  depends  upon  an  enzyme  extractable  from  the  mucosa  by  glycerine, 
and  whose  action  it  seems  follows  Schutz's  law  for  pepsin,  and  the  quantity 
is  as  to  the  square  of  the  enzymotic  products.  This  enzyme,  which  seems 
to  be  produced  from  a  zymogen,  is  very  sensitive  towards  alkalies. 

The  question  whether  the  parietal  cells  principally  or  the  chief  cells, 
or  both,  take  part  in  the  formation  of  free  acid  is  somewhat  disputed.3 
There  can  be  no  doubt  that  the  hydrochloric  acid  of  the  gastric  juice  origi- 
nates from  the  chlorides  of  the  blood,  because,  as  is  well  known,  a  secretion 
of  perfectly  typical  gastric  juice  takes  place  in  the  stomachs  of  fasting  or 

1  Deutsch.  med.  Wochenschr. ,  1899,  and  Pfliiger's  Arch.,  79. 

2  Munch,  med.  Wochenschr.,  1900,  and  Zeitschr.  f.  klin.  Med.,  42,  43.  See  also 
Stade,  Hofmeister's  Beitriige,  3. 

3  See  Heidenhain,  Pfliiger's  Arch.,  18  and  19,  and  Hermann's  Handbuch,  5,  part  I, 
"  Absonderungsvorgiinge  " ;  Klemensiewicz,  Wien.  Sitzungsber.,  71;  Frankel,  Pfliiger's 
Arch.,  48  and  50;  Contejean,  1.  c,  Chapter  II;  Kranenburg,  Archives  Teyler,  Series  II, 
Haarlem,  1901,  and  Mosse,  Centralbl.  f.  Physiol.,  17,  217. 


FORMATION  OF  HYDROCHLORIC  ACID.  307 

starving  animals.  As  the  chlorides  of  the  blood  are  derived  from  the 
food,  it  is  easily  understood,  as  shown  by  Cahn,1  that  in  dogs  after  a  suffi- 
ciently long  common-salt  starvation  the  stomach  secreted  a  gastric  ju'ce 
containing  pepsin,  but  no  free  hydrochloric  acid.  On  the  administration 
of  soluble  chlorides,  a  gastric  juice  containing  hydrochloric  acid  was  imme- 
diately secreted.  On  the  introduction  of  alkali  iodides  or  bromides,  Kulz, 
Xe.wki  and  Schoumow-Simaxowski  2  have  shown  that  the  hydrochloric 
acid  of  the  gastric  juice  is  replaced  by  HBr,  and  to  a  less  extent  by  HI. 
The  secretion  of  free  hydrochloric  acid  from  the  blood  has  been  explained 
in  various  ways,  but  as  yet  there  is  no  satisfactory  theory. 

After  a  full  meal,  when  the  store  of  pepsin  in  the  stomach  is  completely 
exhausted,  Schiff  claims  that  certain  bodies,  especially  dextrin,  have 
the  property  of  causing  a  supply  of  pepsin  in  the  mucous  membrane.  This 
''charge  theory,"  though  experimentally  proved  by  several  investigators, 
nas  nevertheless  not  yet  been  confirmed.  On  the  contrary,  the  state- 
ment of  Schiff  that  a  substance  forming  pepsin,  a  "pepsinogen"  or  "pro- 
pepsin, ' '  occurs  in  the  ventricle  has  been  proved.  Laxgley  3  has  shown, 
positively  the  existence  of  such  a  substance  in  the  mucous  coat.  This 
substance,  propepsin,  shows  a  comparatively  strong  resistance  to  dilute 
alkalies  (a  soda  solution  of  5  p.  m.),  which  easily  destroy  pepsin  (Laxgley). 
Pepsin,  on  the  other  hand,  withstands  better  than  propepsin  the  action  of 
caibon  dioxide,  which  quickly  destroys  the  latter.  The  occurrence  of  a 
rennin  zymogen,  and  possibly  also  a  steapsinogen,  in  the  mucous  coat 
has  been  mentioned  above. 

According  to  Herzex  and  his  collaborators  *  we  must  differentiate  between 
pepsinogens  and  bodies  accelerating  the  flow  of  juice.  To  the  first  belongs  inulin 
and  glycogen,  while  alcohol  belongs  to  the  latter  class  of  bodies.  Dextrin  not 
only  accelerates  the  flow  of  juice,  but  also  acts  as  a  pepsinogen,  especially  as  the 
latter.  Meat  extract  which  has  both  actions  is  especially  a  flow  accelerator. 
The  pepsinogen  action  consists  in  converting  the  zymogen  into  pepsin  and  in 
this  way  increases  the  quantity  of  pepsin;  the  flow-accelerating  substances  in- 
crease the  quantity  of  secretion  of  juice. 

The  question  in  which  cells  the  two  zymogens,  especially  the  propepsin, 
are  produced  has  been  extensively  discussed  for  several  years.  Formerly  it 
was  the  general  opinion  that  the  parietal  cells  were  pepsin  cells,  but  since 
the  investigations  of  Heidexhaix  and  his  pupils,  Laxgley  and  others,  the 
formation  of  pepsin  has  been  shifted  to  the  chief  cells.5 

1  Zeitschr.  f.  physiol.  Chem.,  10. 

2  Kulz,  Zeitschr.  f.  Biologie,  23;  Xencki  and  Schoumow,  Arch,  des  sciences  biol. 
de  St.  Petersbourg,  3. 

'Schiff,  "Lecons  sur  la  physiol.  de  la  digestion,"  1867,  2;  Langley  and  Edkins, 
Journ.  of  Physiol.,  7. 

*  Pfluger's  Arch.,  84. 

*  See  foot-note  3,  page  306. 


308  DIGESTION. 

The  Pyloric  Secretion.  That  part  of  the  pyloric  end  of  the  dog's 
stomach  which  contains  no  fundus  glands  was  dissected  by  Klemensie- 
wicz,  one  end  being  sewed  together  in  the  shape  of  a  blind  sac  and  the 
other  sewed  into  the  stomach.  From  the  fistula  thus  created  he  was  able 
to  obtain  the  pyloric  secretion  of  a  living  animal.  This  secretion  is  alka- 
line, viscous,  jelly-like,  rich  in  mucin,  of  a  specific  gravity  of  1.009-1.010, 
and  containing  16.5-20.5  p.  m.  solids.  It  habitually  contains  pepsin,  which 
has  been  proven  by  Heidenhain  by  observations  on  a  permanent  pyloric 
fistula,  and  the  amount  may  sometimes  be  considerable.  Contejean  has 
investigated  the  pyloric  secretion  in  other  ways,  and  finds  that  it  contains 
both  acid  and  pepsin.  The  alkaline  reaction  of  the  seoretions  investi- 
gated by  Heidenhain  and  Klemensiewicz  is  due,  according  to  Conte 
jean,  to  an  abnormal  secretion  caused  by  the  operation,  because  the  stom- 
ach readily  yields  an  alkaline  juice  instead  of  an  acid  one  under  abnormal 
conditions.  The  statements  of  Heidenhain  and  Klemensiewicz  have 
been  substantiated  by  Akermann,  while  Kresteff,  who  operated  according 
to  another  method,  has  come  to  the  same  results.  Kresteff  l  found  in 
the  juice  (of  dogs)  pepsin,  but  no  chymosin.  Verhaegen  2  has  observed  in 
human  beings  towards  the  end  of  the  ventricle  digestion  a  fluid  not  acid, 
which,  according  to  him,  originates  in  the  pyloric  region. 

The  secretion  of  gastric  juice  under  different  conditions  may  vary  con- 
siderably. The  statements  of  the  quantity  of  gastric  juice  secreted  in  a 
certain  time  are  therefore  so  unreliable  that  they  need  not  be  taken  into 
account. 

The  Chyme  and  the  Digestion  in  the  Stomach.  By  means  of  the  chem- 
ical stimulation  caused  by  the  food,  a  copious  secretion  of  gastric  juice  occurs. 
The  food  is  thereby  freely  mixed  with  liquid  and  is  gradually  converted 
into  a  pulpy  mass  called  chyme.  This  mass  is  acid  in  reaction,  and, 
with  the  exception  of  the  interior  of  large  pieces  of  meat  or  of  other  solid 
foods,  the  chyme  gradually  becomes  acid  throughout.  The  transforma- 
tion products  of  the  digestion  of  proteids  and  carbohydrates  can  be  de- 
tected in  the  chyme;  likewise  more  or  less  changed  undigested  residues  of 
swallowed  food  appear,  which  indeed  form  the  chief  mass  of  the  chyme. 

Since  the  hydrochloric  acid  of  the  gastric  juice  prevents  the  contents  of 
the  stomach  from  fermenting  with  the  generation  of  gas,  those  gases  which 
occur  in  the  stomach  probably  depend,  at  least  in  great  measure,  upon  the 
swallowed  air  and  saliva,  and  upon  those  gases  generated  in  the  intestine 
and  returned  through  the  pyloric  valve.  Planer  found  in  the  stomach- 
gases  of  a  dog  66-68  per  cent  N,  25-33  per  cent  C02,  and  only  a  small 
quantity,  0.8-6.1  per  cent  of  oxygen.     Schierbeck3  has  f-hown  that  a 

'Heidenhain  and  Klemensiewicz,  1.  c. ;    Contejean,  1.  c.,  Chapter  II,  and  Skand. 
Arch.  f.  Physiol.,  6;  Akermann,  ibid.,  5;  Kresteff,  Maly's  Jahresber.,  30. 
2  See  the  work  of  Verhaegen,  La  Cellule,  1896,  1897. 
*  Planer,  Wien.  Sitzungsber.,  42;  Schierbeck,  Skand.  Arch.  f.  Physiol.,  3  and  5. 


GASTRIC  DIGESTION.  30$ 

part  of  th^  carbon  dioxide  is  formed  by  the  mucous  membrane  of  the 
stomach.  The  tension  of  the  carbon  dioxide  in  the  stomach  corresponds, 
according  to  him,  to  30-40  mm.  Hg  in  the  fasting  condition.  It  increases 
after  partaking  food,  independently  of  the  kind  of  food,  and  may  rise  to 
130-140  mm.  Hg  during  dige  tion.  The  curve  of  the  carbon-dioxide 
tension  in  the  stomach  Is  the  same  as  the  curve  of  acidity  in  the  different 
phases  of  digestion,  and  Schierbeck  has  also  found  that  the  carbon-dioxide 
tension  is  considerably  increased  by  pilocarpine,  but  diminished  by  nico- 
tine. According  to  him,  the  carbon  dioxide  of  the  stomach  is  a  product 
of  the  activity  of  the  secretory  cells. 

According  as  the  food  is  finely  or  coarsely  divided  it  passes  sooner  or 
later  through  the  pylorus  into  the  intestine.  From  Busch's  observations 
on  a  human  intestinal  fistula,  it  required  generally  15-30  minutes  after 
eating  for  undigested  food  to  pass  into  the  upper  part  of  the  small  intes- 
tine. In  a  case  of  duodenal  fistula  in  a  human  being  observed  by  Kuhne, 
he  saw,  ten  minutes  after  eating,  uncurdled  but  still  coagulable  milk  and 
small  pieces  of  meat  pass  out  of  the  fistula.  The  time  in  which  the  stomach 
unburdens  itself  of  its  contents  depends,  however,  upon  the  reflex  action 
from  the  stomach  or  intestine  opening  or  closing  the  pylorus,  which  action 
is  dependent  upon  the  quantity  and  character  of  the  food,  the  amount  of 
fat,  and  the  degree  of  acidity  contained  in  the  contents  of  the  stomach 
and  intestine.  The  emptying  of  the  food  into  the  small  intestine  causes, 
as  shown  by  Pawlow,  a  closing  of  the  pylorus  by  chemoreflex  in  which  the 
hydrochloric  acid  and  the  fat  take  part,  and  we  then  find  in  this  regard 
an  alternate  action  between  the  stomach  and  duodenum.1  The  time  neces- 
sary for  the  stomach  to  empty  itself  must  differ  considerably  under  vari- 
ous conditions.  Beaumont  2  found  in  his  extensive  observations  on  the 
Canadian  hunter,  St.  Martin,  that  the  stomach,  as  a  ride,  is  emptied 
1  J-5£  hours  after  a  meal,  depending  upon  the  character  of  the  food. 

The  time  in  which  different  foods  leave  the  stomach  depends  also  upon 
their  digestibility.  In  regard  to  the  unequal  digestibility  in  the  stomach 
of  fo  ids  rich  in  proteids,  which  really  form  the  object  of  the  action  of  the 
gastric  juice,  a  distinction  must  be  m  de  between  the  rapidity  with  which 
the  proteids  are  converted  into  proteoses  and  peptones  and  the  rapidity 
with  which  the  food  is  converted  into  chyme,  or  at  least  so  prepared  that 
it  may  easily  pas?  into  the  intestine.  This  distinction  is  especially  im- 
portant from  a  practical  standpoint.  When  a  proper  food  is  to  be  decided 
upon  in  cases  of  dimini  hed  gastric  digestion,  it  is  important  to  select 
such  foods  as,  independent  of  the  difficulty  or  ease  with  which  their  pro- 

1  Busch,  Virchow's  Arch.,  14;  Kuhne,  Lehrb.  d.  physiol.  Chem.,  53;  (Pawlow  and) 
Serdjukow,  Maly's  Jahresber.,  29;  Lintwarew,  Biochem.  Centralbi.,  1,  Qli.  See  also. 
Richtet,  1.  c. 

2  Beaumont,  "  The  physiology  of  digestion,"  1833. 


310  DIGESTION. 

teid  is  peptonized,  leave  the  stomach  easily  and  quickly,  and  which  require 
as  little  action  as  possible  on  the  part  of  this  organ.  From  this  point  of 
view  those  foods,  as  a  rule,  are  most  digestible  which  are  fluid  from  the 
start  or  may  be  easily  liquefied  in  the  stomach;  but  these  foods  are  not 
always  the  most  digestible  in  the  sens  3  that  their  pro  teid  is  most  easily 
peptonized.  As  an  example,  hard-boiled  white  of  egg  is  more  easily  pep- 
tonized than  fluid  white  of  egg  at  a  degree  of  acidity  of  1-2  p.  m.  HC1 ; l 
nevertheless  we  consider,  and  justly,  that  an  unboiled  or  soft-boiled  egg 
is  easier  to  digest  than  a  hard-boiled  one.  Likewise  uncooked  meat, 
when  it  is  not  chopped  very  fine,  is  not  more  quickly  but  more  slowly 
peptonized  by  the  gastric  juice  than  the  cooked,  but  if  it  is  divided  suffi- 
ciently fine  it  is  often  more  quickly  peptonized  than  th^  cooked. 

The  greater  or  less  facility  with  which  the  different  proteid  foods 
are  digested  in  the  stomach  has  been  comparatively  little  studied.  The 
most  complete  investigations  on  this  subject  are  those  of  Fermi.2  but  as 
they  do  not  allow  of  a  short  discussion  we  must  refer  to  the  original  publi- 
cation. 

As  our  knowledge  of  the  digestibility  of  the  different  foods  in  the 
stomach  is  slight  and  dubious,  so  also  our  knowledge  of  the  action  of  other 
bodies,  such  as  alcoholic  drinks,  bitter  principles,  spices,  etc.,  on  the  nat- 
ural digestion  is  very  uncertain  and  imperfect.  The  difficulties  which 
stand  in  the  way  of  this  kind  of  investigation  are  very  great,  and  therefore 
the  results  obtained  thus  far  are  often  ambiguous  or  conflict  with  each 
other.  For  example,  certain  investigators  have  observed  that  small  quan- 
tities of  alcohol  or  alcoholic  drinks  do  not  prevent  but  rather  facilitate 
digestion;  others  observe  only  a  disturbing  action  while  other  investi- 
gators believe  to  have  found  that  the  alcohol  first  acts  somewhrt  as  a 
disturbing  agent,  but  afterwards,  when  it  is  absorbed,  it  produces  an 
abundant  secretion  of  gastric  juice,  and  thereby  facilitates  digestion  (Gltj- 
zinski,  Chittenden  3).  The  accelerating  action  of  alcohol  upon  the  flow 
of  gastric  juice  has  already  been  mentioned  on  page  303 

The  digestion  of  sundry  foods  is  not  dependent  on  one  organ  alone,  but 
divided  among  several.  For  this  reason  it  is  to  be  expected  that  the  various 
digestive  organs  can  act  for  one  another  to  a  certain  point,  and  that  there- 
fore the  work  of  the  stomach  could  be  taken  up  more  or  less  by  the  intes- 
tine. This  in  fact  is  the  case.  Thus  the  stomachs  of  dogs  and  cats  have 
been  completely  extirpated  or  nearly  so  (Czerny,  Carvallo  ,  and  Pan  hon)  , 
and  also  that  part  necessary  in  the  digestive  process  has  been  eliminated 
by  plugging  the  pyloric  opening  (Ludwig  and  Ogata)  ,  and  in  both  cases  it 

1  Wawrinsky,  Maly's  Jahresber.,  3. 

2  Arch.  f.  (Anat.  u.)  Physiol.,  1901,  Suppld. 

3  Gluzinski,  Deutsch.  Arch.  f.  klin.  Med.,  39;  Chittenden,  Centralbl.  f.  d.  med.  Wis- 
eensch.,  1889;  and  Chittenden  and  Mendel,  and  others,  Amer.  Journ.  of  Physiol.,  1. 


(,'ASTRIC  DIGESTION.  311 

was  possible  to  keep  the  animal  alive,  well  fed,  and  strong  for  B  shorter  or 
longer  time.  This  is  also  true  for  human  beings.1  In  these  cases  it  is 
evident  thai  the  digestive  work  of  the  stomach  was  taken  up  by  the  intes- 
tine:   but  all  food  cannot   be  digested   in   these  cases  to  the  same  extent, 

and  the  connective  tissue  of  meat  especially  is  sometimes  found  to  a  con- 
siderable extent  undigested  in  the  excrements. 

In  order  to  judge  of  the  role  of  the  stomach  in  digestion  the  amount  of 
tin'  digestion  products  in  the  stomach  has  been  determined.  These  deter- 
minations, partly  on  man  and  partly  on  animals,  have  led,  as  is  to  be  expected, 
to  varying  results  (Cahn,  Ellenbergkr  and  Hofmeister,  Chittenden 
and  American).  The  recent  investigations  of  E.  Zunz  2  show  that  boiled 
meat  in  the  stomach  of  a  dog  yields  chiefly  proteoses  with  small  amounts 
of  simpler  cleavage  products,  and  only  very  little  acid  albumin  is  formed. 
The  extent  of  digestion  in  the  stomach  depends  essentially  upon  the  time 
<luring  which  the  food  remains  in  the  organ. 

It  is,  however,  quite  generally  assumed  that  no  peptonization  of  the 
proteids  worth  mentioning  occurs  in  the  stomach,  and  that  the  protein 
foods  are  only  prepared  in  the  stomach  for  the  real  digestive  processes  in 
the  intestine.  That  the  stomach  serves  in  the  first  place  as  a  storeroom 
fellows  from  its  shape,  and  this  function  is  of  special  value  in  certain  new- 
born animals,  for  instance  in  dogs  and  cats.  In  these  animals  the  secretion 
of  the  stomach  contains  only  hydrochloric  acid  but  no  pepsin,  and  the 
casein  of  the  milk  is  converted  by  the  acid  alone  into  solid  lumps  or  a  solid 
coagulum  which  fills  the  stomach.  Small  portions  of  this  coagulum  pass 
into  the  intestine  only  little  by  little,  and  an  overburdening  of  the  intestine 
is  thus  prevented.  In  other  animals,  such  as  the  snake  and  certain  fishes 
which  swallow  their  food  entire,  it  is  certain  that  the  major  part  of  the 
process  of  digestion  takes  place  in  the  stomach.  The  importance  of  the 
stomach  in  digestion  cannot  at  once  be  decided.  It  varies  for  different 
animals,  and  it  may  vary  in  the  same  animal,  depending  upon  the  division 
of  the  food,  the  rapidity  with  which  the  peptonization  takes  place,  the  more 
or  less  rapid  increase  in  the  amount  of  hydrochloric  acid,  and  so  on. 

It  is  a  well-known  fact  that  the  contents  of  the  stomach  may  be  kept 
without  decomposing  for  some  time  by  means  of  hydrochloric  acid,  while, 
on  the  contrary,  when  the  acid  is  neutralized  a  fermentation  commences  by 
which  lactic  acid  and  other  organic  acids  are  formed.  According  to  Cohn 
an  amount  of  hydrochloric  acid  more  than  0.7  p.  m.  completely  arrests 

1  Czerny,  cited  from  Bunge,  Lehrbuch  d.  physiol.  u.  Path.  Chem.,  4.  Aufl.  Thed  2, 
173;  Carvallo  and  Panchon,  Arch.  d.  Physiol.  (5),  7;  Ogata,  Du  Bois-Reymond's 
Arch.,  1883;  Groho,  Arch.  f.  exp.  Path.  u.  Pharm.,  49.  In  regard  to  a  human  case  of 
Schlatter  see  Wr6blewski,  Centralbl.  f.  Physiol.,  11,  665. 

2  Cahn,  Zeitschr.  f.  klin.  Med.,  12;  Ellenberger  and  Hofmeister,  Du  Bois-Reymond's 
Arch.,  1890;  Chittenden  and  Amerman,  Journ.  of  Physiol.,  14;  E.  Zunz,  Hofmeister 's 
Beitrage,  3.     See  also  Reach,  ibid.,  4. 


312  DIGESTION. 

lactic-acid  fermentation,  even  under  otherwise  favorable  circumstances,  and 
according  to  Strauss  and  Bialocour  the  limit  of  lactic-acid  fermentation 
lies  at  1.2  p.  m.  hydrochloric  acid  united  to  organic  bodies.  The  hydro- 
chloric acid  of  the  gastric  juice  has  unquestionably  an  antifermentative 
action,  and  also,  like  dilute  mineral  acids,  an  antiseptic  action.  This  action 
is  of  importance,  as  many  pathogenic  micro-o  ganisms  may  be  destroyed 
by  the  gastric  juice.  The  common  bacillus  of  cholera,  certain  streptococci, 
etc.,  are  killed  by  the  gastric  juice,  while  others,  especially  as  spores,  are 
unacted  upon.  The  fact  that  gastric  juice  can  diminish  or  retard  the 
action  of  certain  toxalbumins,  such  as  tetanotoxin  and  diphtheria  toxin,  is 
also  of  great  interest  (Nencki,  Sieber,  and  Schoumow  :). 

Because  of  this  antifermentative  and  antitoxic  action  of  gastric  juice  it 
is  considered  that  the  chief  importance  of  the  gastric  juice  lies  in  its  anti- 
septic action.  The  fact  that  intestinal  putrefaction  is  not  increased  on  the 
extirpation  of  the  stomach,  as  derived  from  experiments  made  on  man 
and  animals,2  does  not  uphold  this  view. 

In  close  connection  with  the  acid  reaction  of  the  contents  of  the  stomach 
stands  the  question  as  to  the.  extent  of  carbohydrate  digestion  in  this 
organ.  The  salivary  diastase  is  destroyed  by  very  small  quantities  of 
acid,  but  before  a  sufficient  amount  of  hydrochloric  acid  has  collected  to 
destroy  the  action  a  powerful  action  of  saliva  may  go  on  in  the  human 
stomach  and  therefore  sugar  and  dextrin  can  be  readily  detected  in  the 
contents  of  this  organ.  In  carnivora  whose  saliva  has  very  little  dias- 
tatic  action  we  can  ignore  a  priori  any  digestion  of  starch  in  the  stomach 
with  the  exception  of  some  action  of  the  micro-organisms  occurring 
therein.  Friedenthal  3  claims  that  dogs  can  readily  digest  starch,  and 
according  to  him,  the  gastric  juice  of  the  dog  contains  a  diastatic  enzyme 
which  is  even  active  in  a  strong  acid  reaction. 

After  death,  if  the  stomach  still  contains  food,  auto-digestion  goes  on 
not  only  in  the  stomach,  but  also  in  the  neighboring  organs,  during  the 
slow  cooling  of  the  body.  This  leads  to  the  question,  Why  does  the  stomach 
not  digest  itself  during  life?  Ever  since  Pavy  has  shown  that  after  tying 
the  smaller  blood-vessels  of  the  stomach  of  dogs  the  corresponding  part  of 
the  mucous  membrane  was  digested,  efforts  have  been  made  to  find  the 
cause  in  the  neutralization  of  the  acid  of  the  gastric  juice  by  the  alkali  of 
the  blood.  That  the  reason  for  the  non-digestion  during  life  is  to  be  sought 
for  in  the  normal  circulation  of  the  blood  cannot  be  contradicted;  but  the 

1  Cohn,  Zeitschr.  f.  physiol.  Chem.,  14;  Strauss  and  Bialocour,  Zeitschr.  f.  klin. 
Med,  28.  See  also  Kiihne,  Lehrb.,  57;  Bunge,  Lehrb.  d.  Physiol.,  4,  Aufl.  148  and 
159;  Hirschfeld,  Pfluger's  Arch.,  47;  Nencki,  Sieber,  and  Schoumow,  Centralbl.  f. 
Bacteriol.,  etc.,  23.  In  regard  to  the  action  of  gastric  juice  upon  pathogenic  microbes 
we  must  refer  the  reader  to  the  handbooks  of  bacteriology. 

2  See  Carvallo  and  Panchon,  1.  c,  and  Schlatter  in  Wroblewski,  1.  c. 

3  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl. 


EXAMINATION  OF   THE  GASTRIC  CONTENTS.  313 

reason  is  not  to  be  found  in  the  neutralization  of  the  acid.  The  investi- 
gations of  Fermi,  Mathes,  and  Otte  x  show  that  the  blood  circulation  acts 
in  an  indirect  manner  by  the  normal  nourishment  of  the  cell  protoplasm, 
and  this  is  the  reason  why  the  digestive  fluids,  the  gastric  juice  as  well  as 
the  pancreatic  juice,  act  differently  upon  the  living  protoplasm  as  com- 
pared to  the  dead.  We  know  nothing  about  this  resistance  of  the  living 
protoplasm,  but  perhaps  it  stands  in  close  connection  with  the  secretion 
of  antipepsin  (see  page  299)  discovered  by  Danilewsky  and  Weixlaxd. 

Under  pathological  conditions  irregularities  in  the  secretion  as  well  as  in 
the  absorption  and  in  the  mechanical  work  of  the  stomach  may  occur.  Pepsin 
is  almost  always  present,  although  the  amount  may  vary  considerably,  but  the 
absence  of  the  rennin,  as  above  stated,  may  occur  in  many  cases.  In  regard 
to  the  acid  we  must  remark  that  the  secretion  is  sometimes  increased  so  that 
an  abnormally  acid  gastric  juice  is  secreted  and  at  other  times  it  may  be  dimin- 
ished so  that  little  if  any  hydrochloric  acid  is  formed.  A  hypersecretion  of 
acid  gastric  juice  sometimes  occurs.  In  the  secretion  of  too  little  hydrochloric 
acid  the  same  conditions  appear  as  after  the  neutralization  of  the  acid  contents 
of  the  stomach  outside  of  the  organism.  Fermentation  processes  now  appear  in 
which,  besides  lactic  acid,  there  appear  also  volatile  fatty  acids,  such  as  butyric  and 
acetic  acids,  etc.,  and  gases  like  hydrogen.  These  fermentation  products  are  there- 
fore often  found  in  the  stomach  in  cases  of  chronic  catarrh  of  the  stomach,  which 
may  give  rise  to  belching,  pyrosis,  and  other  symptoms. 

Among  the  foreign  substances  found  in  the  contents  of  the  stomach  we  have 
urea,  or  ammonium  carbonate  derived  therefrom  in  uraemia;  blood,  which 
generally  forms  a  dark-brown  mass  through  the  presence  of  haematin,  due  to 
the  action  of  the  gastric  juice;  bile,  which,  especially  during  vomiting,  easily 
finds  its  way  through  the  pylorus  into  the  stomach,  but  whose  presence  seems 
to  be  without  importance. 

If  it  is  desired  to  test  the  gastric  juice  or  the  contents  of  the  stomach 
for  pepsin,  fibrin  may  be  employed.  If  this  is  thoroughly  washed  immedi- 
ately after  beating  the  blood,  well  pressed,  and  placed  in  glycerine,  it  may  be 
kept  in  serviceable  condition  for  an  indefinitely  long  time.  The  gastric  juice 
or  the  contents  of  the  stomach — the  latter,  if  necessary,  having  been  pre- 
viously diluted  with  1  p.  m.  hydrocholoric  acid — is  filtered  and  tested  with 
fibrin  at  ordinary  temperature.  (It  must  not  be  forgotten  that  a  control 
test  must  be  made  with  acid  alone  and  another  portion  of  the  same  fibrin.) 
If  the  fibrin  Is  not  noticeably  digested  within  one  or  two  hours,  no  pepsin  is 
present,  or  at  most  there  are  only  slight  traces. 

In  testing  for  rennin  the  liquid  must  be  first  carefully  neutralized.  To 
10  c.  c.  of  unboiled,  amphoteric  (not  acid)  cow's  milk  add  1-2  c.  c.  of  the 
filtered  neutralized  liquid.  In  the  presence  of  rennin  the  milk  should 
coagulate  to  a  solid  mass  at  the  temperature  of  the  body  in  the  course  of 
10-12  minutes  without  changing  its  reaction.  If  the  milk  is  diluted  too 
much  by  the  addition  of  the  liquid  of  the  stomach,  only  coarse  flakes  are 
obtained  and  no  solid  coagulum.  Addition  of  lime-salts  is  to  be  avoided, 
as  in  great  excess  they  may  produce  a  partial  coagulation  even  in  the 
absence  of  typical  rennin. 

1  Pa-vy,  Phil.  Transactions,  158,  Part  I,  and  Guy's  Hospital  Reports,  13;  Otte 
Travaux  du  laboratoire  de  l'Institut  de  Physiol,  de  Liege,  5,  1S90,  which  also  contains 
the  literature. 


314  DIGESTION. 

In  many  cases  it  is  especially  important  to  determine  the  degree  of 
acidi  y  of  the  gastric  juice.  This  may  be  done  by  the  ordinary  titration 
methods.  Phenolphthalein  must  not  be  used  as  an  indicator,  as  too  high 
results  are  produced  in  the  presence  of  large  quantities  of  proteids.  Good 
results  may  be  obtained,  on  the  contrary,  by  using  very  delicate  litmus 
paper.  As  the  acid  reaction  of  the  contents  of  the  stomach  may  be  caused 
simultaneously  by  several  acids,  still  the  degree  of  acidity  is  here,  as  in 
other  cases,  expressed  in  only  one  acid,  e.g.,  HC1.  Generally  the  acidity 
is  designated  by  the  number  of  c.  c.  of  N/10  caustic  soda  which  is  required 
to  neutralize  the  several  acids  in  100  c.  c.  of  the  liquid  of  the  stomach. 
An  acidity  of  43  per  cent  means  that  100  c.  c.  of  the  liquid  of  the 
stomach  required  43  c.  c.  of  N/10  caustic  soda  to  neutralize  it. 

The  acid  reaction  may  be  partly  due  to  free  acid,  partly  to  acid  salts 
(monophosphates),  and  partly  to  both.  According  to  Leo1  one  can  test 
for  acid  phosphates  by  calcium  carbonate,  which  is  not  neutralized  there- 
with, while  the  free  acids  are.  If  the  gastric  content  has  a  neutral  reaction 
after  shaking  with  calcium  carbonate  and  the  carbon  dioxide  is  driven  out 
by  a  current  of  air,  then  it  contains  only  free  acid;  if  it  has  an  acid  reaction, 
then  acid  phosphates  are  present,  and  if  it  is  less  acid  than  before,  it  con- 
tains both  free  acid  and  acid  phosphate.  This  method  can  also  be  applied 
in  the  estimation  of  free  acid. 

It  is  also  important  to  be  able  to  ascertain  the  nature  of  the  acid  or 
acids  occurring  in  the  contents  of  the  stomach.  For  this  purpose,  and 
especially  for  the  detection  of  free  hydrochloric  acid,  a  great  number  of  color 
reactions  have  been  proposed  which  are  all  based  upon  the  fact  that  the 
coloring  substance  gives  a  characteristic  color  with  very  small  quantities 
of  hydrochloric  acid,  while  lactic  acid  and  the  other  organic  acids  do  not 
give  these  colorations,  or  only  in  a  certain  concentration,  which  can  hardly 
exist  in  the  contents  of  the  stomach.  These  reagents  are  a  mixture  of 
ferric  acetate  and  potassium  sulphocyanide  solution  (Mohr 's  reagent 
has  been  modified  by  several  investigators),  methylanilin-violet,  tro- 
p^eolin  00,  Congo  red,  malachite-green,  phloroglucin-vanillin,  di- 
methylaminoazobenzene,  and  others.  As  reagents  for  free  lactic  acid 
Uffelmann  suggests  a  strongly  diluted,  amethyst-blue  solution  of  ferric 
chloride  and  carbolic  acid  or  a  strongly  diluted,  nearly  colorless  solu- 
tion of  ferric  chloride.  These  give  a  yellow  color  with  lactic  acid,  but 
not  with  hydrochloric  acid  or  with  volatile  fatty  acids. 

The  value  of  these  reagents  in  testing  for  free  hydrochloric  acid  or  lactic 
acid  is  still  disputed.  Among  the  reagents  for  free  hydrochloric  acid 
Gunzburg's  test  with  phloroglucin-vanillin,  and  the  test  with  tropseolin  00, 
performed  in  moderate  heat  as  suggested  by  Boas,  and  the  test  with 
dimethylaminoazobenzene,  which  is  the  most  delicate,  seem  to  be  the  most 
valuable.  If  these  tests  give  positive  results,  then  the  presence  of  hydro- 
chloric acid  may  be  considered  as  proved.  A  negative  result  does  not 
eliminate  the  presence  of  hydrochloric  acid,  as  the  delicacy  of  these  reac- 
tions has  a  limit,  and  also  the  simultaneous  presence  of  proteid,  peptones, 
and  other  bodies  influences  the  reactions  more  or  less.  The  reactions  for 
lactic  acid  may  also  give  negative  results  in  the  presence  of  comparatively 
large  quantities  of  hydrochloric  acid  in  the  liquid  to  be  tested.     Sugar, 

1  Centralbl.  f.  d.  med.  Wissensch.,  1889,  481,  and  Pfliiger's  Arch.,  48,  614. 


EXAMINATION  OF   THE  GASTRIC  CONTENTS.     '  315 

Blllphooyanides,  and  other  bodies  may  act  with  these  reagents  similarly  to 
lactic  acid. 

In  testing  for  lactic  acid  it  is  safest  to  shake  the  material  with  ether  and 
test  the  residue  after  the  evaporation  of  the  solvent,  <  m  the  evaporation  of 
the  ether  the  residue  may  be  tested  in  several  ways.  BOAS  '  utilizes  the 
property  possessed  l»y  lactic  acid  of  being  oxidized  into  aldehyde  and 
formic  acid  on  careful  oxidation  with  sulphuric  acid  and  manganese  dioxide. 
The  aldehyde  is  detected  by  its  forming  iodoform  with  an  alkaline  iodine  solu- 
tion or  by  its  forming  aldehyde  mercury  with  Nesslbr's  reagent.  The  quan- 
titative estimation  consists  in  the  formation  of  iodoform  with  N/10  iodine 
solution  and  caustic  potash,  adding  an  excess  of  hydrochloric  acid  and  titrat- 
ing with  a  N  10 sodium  arsenite solution,  and  retitrating  with  iodine  solution, 
after  the  addition  of  starch-paste,  until  a  blue  coloration  is  obtained.  This 
method  presupposes  the  use  of  ether  entirely  free  from  alcohol.  (See  the 
original.) 

In  order  to  be  able  to  correctly  judge  of  the  value  of  the  different 
reagents  for  free  hydrochloric  acid,  it  is  naturally  of  greatest  importance  to 
be  clear  in  regard  to  what  we  mean  by  free  hydrochloric  acid.  It  is  a  well- 
known  fact  that  hydrochloric  acid  combines  with  proteids,  and  a  consider- 
able part  of  the  hydrochloric  acid  may  therefore  exist  in  the  contents  of 
the  stomach,  after  a  meal  rich  in  proteids,  in  combination  with  proteids. 
This  hydrochloric  acid  combined  with  proteids  cannot  be  considered  as  free, 
and  it  is  for  this  reason  that  certain  investigators  consider  such  methods  as 
that  of  Sjoqvist,  which  will  be  described  below,  as  of  little  value.  How- 
ever, it  must  be  remarked  that,  according  to  the  unanimous  experience  of 
many  investigators,  the  hydrochloric  acid  combined  with  proteids  is  physio- 
logically active.  Those  reactions  (color  reactions)  which  only  respond  to 
actually  free  hydrochloric  acid  do  not  show  the  physiologically  active 
hydrocholric  acid.  The  suggestion  of  determining  the  "physiologically 
active"  hydrochloric  acid  instead  of  the  "free"  seems  to  be  correct  in  prin- 
ciple; and  as  the  conceptions  of  free  and  of  physiologically  active  hydro- 
chloric acid  are  not  the  same  it  must  always  be  well  defined  whether  one 
wishes  to  determine  the  actually  free  or  the  physiologically  active  hydro- 
chloric acid  before  it  is  possible  of  the  value  of  a  certain  reaction. 

Various  titration  methods  have  been  suggested  for  the  estimation  of 
the  free  hydrochloric  acid,  but  these  cannot  yield  conclusive  results  for 
the  reasons  given  in  a  previous  chapter  (see  estimation  of  the  alkalinity 
of  the  blood-serum,  page  160).  For  this  determination  physico-chemical 
methods  are  necessary,  but  they  have  not  been  used  to  any  great  extent 
for  clinical  purposes  on  account  of  the  difficulty  in  their  manipulation. 
As  it  is  not  within  the  scope  of  this  book  to  give  the  various  methods  for 
the  quantitative  estimation  of  hydrochloric  acid  for  clinical  purposes  we 
must  refer  to  the  various  handbooks  for  clinical  methods,  such  as  those 
of  v.  Jaksch,  Eulenberg,  Kolle  and  Weixtraud,  and  the  work  of  O. 
Rkissxer,2  for  details  as  to  the  qualitative  and  quantitative  tests  for 
hydrochloric  acid  and  lactic  acid. 

1  Deutsch.  med.  Wochenschr.,  1893,  and  Miinchener  med.  Wochenschr.,  1S93. 
*  Zeitschr.  f.  klin.  Med.,  48. 


316  DIGESTION. 

The  methods  suggested  by  Leo,  Hayem  and  Winther,  Martins  and 
Luttke,  and  by  Reissner,  as  well  as  the  following  method  of  Morner 
and  Sjoqvist,1  are  used  for  the  quantitative  estimation  of  the  total  hydro- 
chloric acid. 

The  method  of  K.  Morner  and  Sjoqvist  depends  on  the  following  principle: 
When  the  gastric  juice  is  evaporated  to  dryness  with  barium  carbonate  and  then 
calcined  the  organic  acids  burn  up  and  give  insoluble  barium  carbonate,  while 
the  hydrochloric  acid  forms  soluble  barium  chloride.  From  the  quantity  of 
this  the  original  amount  of  hydrochloric  acid  can  be  calculated.  10  c.  c.  of  the 
filtered  contents  of  the  stomach  is  mixed  in  a  small  platinum  or  silver  dish  with  a 
knife-point  of  barium  carbonate  free  from  chlorides  and  evaporated  to  dryness. 
The  residue  is  burnt  and  allowed  to  glow  for  a  few  minutes.  The  cooled  carbon 
is  gently  rubbed  with  water  and  completely  extracted  with  boiling  water  and 
the  filtrate  (about  50  c.  c.)  precipitated  by  ammonium  chromate  after  the  addi- 
tion of  ammonium  acetate  and  acetic  acid  and  boiling.  The  carefully  collected 
precipitate  is  washed  and  dissolved  in  water  by  the  aid  of  a  little  HC1,  KI,  and  hy- 
drochloric acid  added  and  titrated  with  hyposulphite  solution.  The  reactions  take 
place  as  follows:  4HCl+2BaC03=2BaC^+2HaO+2C02;  2BaCl2+2(NH4)2Cr04  = 
2BaCr04+  4NH4C1 ;  2BaCr04+  16HC1+  6KI  =  2BaCl2+  Cr2Cl6+  8H20+  6KC1+  3I2 ; 
and  3I2+ ONa^SaOg  =  6NaI+ 3Na2S406.  Each  cubic  centimeter  of  the  hyposul- 
phite corresponds  to  3  mgm.  HC1.  Complete  directions  for  the  necessary  solu- 
tions and  for  the  performance  of  the  method  may  be  found  in  Sjoqvist, 
Zeitschr.  f.  klin.  Med.,  32. 

In  testing  for  volatile,  fatty  acids  the  contents  of  the  stomach  should  not 
be  directly  distilled,  as  volatile  fatty  acids  may  be  formed  by  the  decom- 
position of  other  bodies,  such  as  proteid  and  haemoglobin.  The  neutral- 
ized contents  of  the  stomach  are  therefore  precipitated  with  alcohol  at 
ordinary  temperature,  filtered  quickly,  pressed,  and  repeatedly  extracted 
with  alcohol.  The  alcoholic  extracts  are  made  faintly  alkaline  by  soda 
and  the  alcohol  distilled.  The  residue  is  now  acidified  by  sulphuric  or 
phosphoric  acid  and  distilled.  The  distillate  is  neutralized  by  soda  and 
evaporated  to  dryness  on  the  water-bath.  The  residue  is  extracted  with 
absolute  alcohol,  filtered,  the  alcohol  distilled  off,  and  this  residue  dissolved 
in  a  little  water.  This  solution  may  be  directly  tested  for  acetic  acid  with 
sulphuric  acid  and  alcohol  or  with  ferric  chloride.  Formic  acid  may  be 
tested  for  by  silver  nitrate,  which  quickly  gives  a  black  precipitate;  and 
butyric  acid  is  detected  by  the  odor  after  the  addition  of  an  acid.  In 
regard  to  the  methods  for  more  fully  investigating  the  different  volatile 
fatty  acids,  the  reader  is  referred  to  other  text-books. 

III.  The  Glands  of  the  Mucous  Membrane  of  the   Intestine  and 

their  Secretions. 

The  Secretion  of  Brunner's  Glands.  These  glands  are  partly  considered 
as  small  pancreatic  glands  and  partly  as  mucous  or  salivary  glands.  Their 
importance  in  various  animals  is  different.  According  to  Grutzner  they 
are  closely  related  in  dogs  to  the  pyloric  glands  and  contain  pepsin.  This 
also  coincides  with  the  observations  of  Glaessner  and  of  Ponomarew,3 

1  In  regard  to  the  methods  here  mentioned  see  Reissner,  1.  c. 

2  Grutzner,  Pfl  tiger's  Arch.,  12;  Glaessner,  Hofmeister's  Breitage,  1;  Ponomarew, 
Biochem.  Centralbl.,  1,  351. 


INTESTINAL  JUICE.  317 

which  differ  from  each  other  only  in  that  Ponomarew  finds  that  the  secre- 
tion is  inactive  in  alkaline  reaction  and  only  contains  pepsin,  while  Glaessxkk 
claims  it  is  active  in  both  acid  and  alkaline  reaction  and  that  it  contains 
propepsin.  The  statements  as  to  the  occurrence  of  a  diastatic  enzyme 
are  disputed. 

The  Secretion  of  Lieberkuhn's  Glands.  The  secretion  of  these  glands 
has  been  studied  by  the  aid  of  a  fistula  in  the  intestine  according  to  the 
method  of  Thiry  and  Vella.  Very  little  if  any  secretion  takes  place  in 
fasting  animals  (dogs)  when  the  mucous  membrane  is  not  irritated.  In 
lambs  Pregl  found  the  secretion  continuous.  The  ingestion  of  food 
causes  a  secretion,  and  in  lambs  increases  the  secretion  already  taking 
place.  Mechanical,  chemical,  and  electrical  stimulants  act  in  the  same 
manner  in  dogs  (Thiry).  The  secretion  is  also  markedly  increased  in 
man  by  the  local  irritation  of  the  mucous  membrane  (Hamburger  and 
Hekma1).  In  the  cases  observed  by  these  experimenters  the  flow  of 
fluid  was  greatest  at  night  as  well  as  between  five  and  eight  o'clock  in 
the  afternoon,  and  was  lowest  between  two  and  five  o  'clock  in  the  after- 
noon. Pilocarpine  does  not  increase  the  secretion  in  lambs,  and  in  dogs 
it  does  not  seem  to  be  always  active  (Gamgee  2).  The  quantity  of  this 
secretion  in  the  course  of  twenty-four  hours  has  not  been  exactly  deter- 
mined. 

In  the  upper  part  of  the  small  intestine  of  the  dog  this  secretion  is 
scanty,  slimy,  and  gelatinous ;  in  the  lower  part  it  is  more  fluid,  with  gelat- 
inous lumps  or  flakes  (Rohmann).  Intestinal  juice  has  a  strong  alkaline 
reaction  towards  litmus,  generates  carbon  dioxide  on  the  addition  of  an 
acid,  and  contains  (in  dogs)  nearly  a  constant  quantity  of  NaCl  and 
NajCOj,  4.8-5  and  4-5  p.  m.  respectively  (Gumilewski,  Rohmann  3). 
The  intestinal  juice  of  the  lamb  corresponded  to  an  alkalinity  of  4.54  p.  m. 
Na2C03.  It  contains  proteid  (Thiry  found  8.01  p.  m.),  the  quantity 
decreasing  with  the  duration  of  the  elimination.  The  quantity  of  solids 
varies.  In  dogs  the  quantity  of  solids  is  12.2-24.1  p.  m.  and  in  lambs 
29.S5  p.  m.  The  specific  gravity  of  the  intestinal  juice  of  the  dog,  accord- 
ing to  the  observations  of  Thiry,  is  1.010-1.0107,  and  in  lambs  1.01427 
(Pregl).  The  intestinal  juice  from  lambs  contains  1S.097  p.  m.  proteid, 
1.274  p.  m.  proteoses  and  mucin,  2.29  p.  m.  urea,  and  3.13  p.  m.  remain- 
ing organic  bodies. 

We  have  the  investigations  of  Demant,  Turby  and  Manning,  H.  Ham- 

1  Thiry,  Wien.  Sitzungsber.,  50;  Vella,  Moleschott's  Untersuch.,  13;  Pregl,  Pfliiger's 
Arch.,  61;  Gamgee,  Physiol.  Chem.,  2,  410,  where  Vella  and  Masloff  are  quoted; 
Kniger,  Zeitschr.  f.  Biologie,  3";  Hamburger  and  Hekma,  Journ.  de  Physiol.,  4. 

1  Gamgee,  1.  c. 

'Gumilewski,  Pfliiger's  Arch.,  39;  Rohmann,  ibid.,  41.  ' 


318  DIGESTION. 

burger  and  Hekma  and  Nagano  *  on  the  human  intestinal  juice.  Human 
intestinal  juice  has  a  low  specific  gravity,  nearly  1.007,  about  10-14  p.  m. 
solids,  and  is  strongly  alkaline  towards  litmus.  The  content  of  alkali  calcu- 
lated as  sodium  carbonate  is  2.2  p.  m.,  according  to  Nagano,  Hamburger 
and  Hekma,  and  5.8-6.7  p.  m.  NaCl.  The  determination  of  the  freezing- 
point  resulted  —0.62°  (Hamburger  and  Hekma). 

In  regard  to  the  enzyme  content  opinions  are  unanimous  that  the  juice  of 
animals  as  well  as  of  man  has  no  fat-splitting  or  proteid  solvent  action,  while 
it  has  a  very  faint  amylolytic  action.  The  juice,  and  to  a  high  degree  also  the 
mucous  coat,  contain  invertase  and  maltase,  which  fact  has  been  recently 
substantiated  by  the  observations  of  Paschutin,  Brown  and  Heron, 
Bastianelli  and  Tebb.2  A  lactose-inverting  enzyme,  a  lactase,  also 
occurs,  as  shown  by  Rohmann  and  Lappe,  Pautz  and  Vogel,  Weinland  , 
and  Orban,3  in  new-born  infants  and  young  animals  and  also  in  grown 
mammals  who  were  fed  upon  a  milk  diet.  The  lactase  is  found  to  a 
greater  extent  in  the  mucosa  than  in  the  juice. 

The  intestinal  juice,  as  above  stated,  contains  no  proteid-digesting 
enzyme  in  the  ordinary  sense,  at  least  in  any  appreciable  amounts.  On 
the  contrary  it  contains  another  enzyme  which  has  a  proteolytic  action 
called  erepsin. 

Erepsin.  This  enzyme,  discovered  by  0.  Cohnheim,  has  no  direct 
action  upon  native  proteids  with  the  exception  of  casein,  but  has  the  power 
of  splitting  proteoses  and  peptones.  In  this  change  mono  as  well  as  diamino 
acids  are  produced,  but  this  action  differs  from  autolysis  by  only  yielding 
little  ammonia.  Erepsin,  which  must  not  be  confounded  with  trypsin  or  with 
enterokinase,  which  will  be  spoken  of  later,  occurs  in  the  intestinal  juice 
of  man  (Hamburger  and  Hekma)  as  well  as  in  the  dog  (Salaskin).  The 
quantity  of  erepsin  secreted  seems,  according  to  Salaskin,  Kutscher  and 
Seemann,4  to  be  only  very  small,  while  the  mucous  coat  itself  is  richer 
therein,  and  this  enzyme  probably  therefore  has  principally  an  intracellular 
action.     Erepsin  becomes  inactive  on  heating  to  59°  C. 

Besides  erepsin  and  the  other  enzymes  mentioned  the  intestinal  mucosa 
also  contains  antienzymes,  antipepsin  and  antitrypsin  (Danilewsky  and 


1  Demant,  Virchow's  Arch.,  75;  Turby  and  Manning,  Centralbl.  f.  d.  med.  Wis- 
senschft.,  1892,  945;  Hamburger  and  Hekma,  1.  c;  Nagano,  Mitt,  aus  d.  Grenzgeb. 
d.  Med.  u.  Chir.,  9. 

2  Paschutin,  Centralbl.  f.  d.  med.  Wissensch.,  1870,  561;  Brown  and  Heron,  Annal. 
d.  Chem.  u.  Pharm.,  204;  Bastianelli,  Moleschott's  Untersuch.  zur  Naturlehre,  14. 
This  contains  all  the  older  literature.  See  also  Miura,  Zeitschr.  f.  Biologie,  32;  Wid- 
dicombe,  Journ.  of  Physiol.,  28;  Tebb,  ibid.,  15. 

3  Rohmann  and  Lappe,  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  Pautz  and  Vogel, 
Zeitschr.  f.  Biologie,  32;  Weinland,  ibid.,  38;  Orban,  Maly's  Jahresber.,  29. 

♦Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  33,  35.  36;  Salaskin,  ibid.,  35;  Kutscher 
and  Seemann,  ibid.,  35;  Hamburger  and  Hekma.  1.  c. 


THE  PANCREAS.  310 

Weinland,1  also  enierokinase  or  a  mother-substance  of  tho  same,  and 
finally  also  the  so-called  prosecretin.  These  two  last-mentioned  bodies, 
which  are  closely  connected  with  the  secretion  of  pancreatic  juice,  will 
be  discussed  in  connection  with  tlii -  digestive  fluid. 

The  secretion  of  the  glands  in  the  large  intestine  seems  to  consist  chiefly 
of  mucus.  Fistulas  have  also  been  introduced  into  these  parts  of  the 
intestine,  which  are  chiefly  if  not  entirely  to  be  considered  as  absorption 
organs.  Th-1  investigations  on  the  action  of  this  secretion  on  nutritive 
bodies  have  not  as  yet  yielded  any  positive  results. 

IV.    The   Pancreas  and   Pancreatic  Juice. 

In  invertebrates,  which  have  no  pepsin  digestion  and  which  also  have 
no  formation  of  bile,  the  pancreas,  or  at  least  an  analogous  organ,  seems  to 
be  the  essential  digestive  gland.  On  the  contrary,  an  anatomically  charac- 
teristic pancreas  is  absent  in  certain  vertebrates  and  in  certain  fishes. 
Those  functions  which  should  be  regulated  by  this  organ  seem  to  be  per- 
formed in  these  animals  by  the  liver,  which  may  be  rightly  called  the  HEPA- 
topancreas.  In  man  and  in  most  vertebrates  the  formation  of  bile  and  of 
certain  secretions  containing  enzymes  important  for  digestion  is  divided 
between  the  two  organs,  the  liver  and  the  pancreas. 

The  pancreatic  gland  is  similar  in  certain  respects  to  the  parotid  gland. 
The  secreting  elements  of  the  former  consist  of  nucleated  cells  whose  basis 
forms  a  mass  rich  in  proteids,  which  expands  in  water  and  in  which  two 
distinct  zones  exist.  The  outer  zone  is  more  homogeneous,  the  inner  cloudy. 
due  to  a  quantity  of  granules.  The  nucleus  lies  about  midway  between  the 
two  zones,  but  this  position  may  change  with  the  varying  relative  size  of 
the  two  zones.  According  to  Heidexhaix  2  the  inner  part  of  the  cells 
diminishes  in  size  during  the  first  stages  of  digestion,  in  which  the  secretion 
i-  active,  while  at  the  same  time  the  outer  zone  enlarges  owing  to  the  ab- 
sorption of  new  material.  In  the  later  stage,  when  the  secretion  has 
decreased  and  the  absorption  of  the  nutritive  bodies  has  taken  place,  the 
inner  zone  enlarges  at  the  expense  of  the  outer,  the  substance  of  the  latter 
having  been  converted  into  that  of  the  former.  Under  physiological  con- 
ditions the  glandular  cells  are  undergoing  a  constant  change,  at  one  time 
consuming  from  the  inner  part  and  at  another  time  growing  from  the  outer 
part.  The  inner  granular  zone  is  converted  into  the  secretion,  and  the 
outer,  more  homogeneous  zone,  which  contains  the  repairing  material,  is 
then  converted  into  the  granular  substance.  The  so-called  islands  of 
Laxgerhaxs  are  related  to  the  internal  secretion  or  contain  a  substance 
taking  part  in  the  transformation  of  the  sugar  of  the  animal  body. 

1  See  foot-note  6,  page  299.  '  Pfliiger's  Arch.,  10. 


320  DIGESTION. 

The  chief  portion  of  protein  substances  contained  in  the  gland  consists, 
it  seems,  of  nucleoproteids,  while  the  globulins  and  albumins  occur  only  to  a 
slight  extent  as  compared  to  the  nucleoproteids.  Among  the  compound 
proteids  is  the  substance  studied  and  isolated  by  Umber  but  previously  dis- 
covered by  Hammarsten  x  and  called  a-proteid.  This  nucleoproteid  con- 
tains, as  an  average,  1.67  per  cent  P.  1.29  per  cent  S,  17.12  per  cent  N, 
and  0.13  per  cent  Fe.  It  yields  on  boiling  /3-proteid,  so  called  by  Hammar- 
sten, and  which  is  much  richer  in  phosphorus  than  the  nucleoproteid. 
The  native  proteid  (a)  is  the  mother-substance  of  guanylic  acid;  accord- 
ing to  Umber  it  dissolves  by  pepsin  digestion  without  leaving  any  residue 
and  yields  on  trypsin  digestion  guanylic  acid  on  one  side  and  proteoses 
and  peptones  on  the  other.  It  can  be  extracted  from  the  gland  by  a  physio- 
logical salt  solution  and  is  precipitated  by  acetic  acid.  Besides  this  com- 
pound proteid  the  pancreas  must  contain  at  least  one  other,  which  is  the 
mother-substance  of  the  thymonucleic  acid  obtainable  from  the  pancreas. 

Besides  these  protein  substances  the  gland  contains  also  several  enzymes, 
or  more  correctly  zymogens,  which  will  be  discussed  later.  Among  the 
extractive  bodies,  which  are  probably  in  part  formed  by  post-mortem 
changes  and  chemical  action,  we  must  mention  leucin  (butalanin),  tyrosin, 
purin  bases  in  variable  quantities,2  inosite,  lactic  acid,  volatile  fatty  acids,  and 
fats.  The  mineral  bodies  vary  considerably  in  quantity  not  only  in  animals 
and  man  but  also  in  men  and  women  (Gossmann).  The  calcium  seems, 
according  to  Gossmann,  to  exist  in  much  greater  amount  than  the  mag- 
nesium. According  to  the  investigations  of  Oidtmann  the  pancreas  of 
an  old  woman  contains  745.3  p.  m.  water,  245.7  p.  m.  organic,  and  9.5  p.  m. 
inorganic  substances.  Gossmann  3  found  in  a  man  17.92  p.  m.  ash  and 
13.05  p.  m.  in  a  woman. 

Besides  the  already-mentioned  (Chapter  VIII)  relationship  to  the  trans- 
formation of  sugar  in  the  animal  body,  the  pancreas  has  the  property  of 
secreting  a  juice  especially  important  in  digestion. 

Pancreatic  Juice.  This  secretion  may  be  obtained  by  adjusting  a  fistula 
in  the  excretory  duct,  according  to  the  methods  suggested  by  Bernard, 
Ludwig,  and  Heidenhain,  and  perfected  by  Pawlow.4  If  the  operation 
is  performed  with  sufficient  rapidity  and  under  favorable  conditions  a 
powerfully  active  secretion  may  be  obtained  either  immediately  after  the 
operation  (temporary  fistula)  or  after  some  time  (permanent  fistula). 

1  Umber,  Zeitschr.  f.  klin.  Med.,  40  and  43;  Hammarsten,  Zeitschr.  £.  physiol. 
Chem.,  19. 

2  See  Kossel,  ibid.,  8. 

3  Gossmann,  Maly's  Jahresber.,  30;  Oidtmann,  cited  from  Gorup-Besanez,  Lehr- 
buch,  4  Ed.,  732. 

4  Bernard,  Lecons  de  Physiol.,  2, 190;  Ludwig,  see  Bernstein,  Arbeiten  a  d.  physiol. 
Anstalt  zu  Leipzig,  1869;  Heidenhain,  Pfliiger's  Arch.,  10,  604;  Pawlow,  Die  Arbeit 
der  Verdauungsdriisen,  Wiesbaden,  1898,  and  Ergebnisse  der  Physiologie,  1,  Abt.  I. 


PANCREATIC  JUICE.  321 

In  herbivora.  such  as  rabbits,  whose  digestion  is  uninterrupted,  the 
secretion  of  the  pancreatic  juice  is  continuous.  In  carnivora  it  seems,  en 
the  contrary,  to  be  intermittent  and  dependent  on  the  digestion.  During 
starvation  the  secretion  almost  stops,  but  commences  again  after  partaking 
of  food  and  reaches  its  maximum,  according  to  Bernstein,  Heidenhain,  and 
others,  within  the  first  three  hours.  According  to  Pawlow  and  his  school 
(  Walther  ')  this  maximum  is  dependent  upon  the  character  of  the  food. 
With  milk  diet  it  appears  within  three  to  four  hours,  after  bread  diet  at 
the  end  of  the  second  hour,  and  with  a  meat  diet  it  arrives  still  sooner. 
The  quality  of  the  juice  is  also,  according  to  Pawlow 's  school,  dependent 
upon  the  food,  and  the  amount  of  the  three  enzymes,  diastase,  trypsin,  and 
steapsin,  changes  with  the  variety  of  food.  The  observations  which  form 
the  basis  of  this  view  have  been  somewhat  differently  explained  in  light 
of  recent  investigations. 

It  has  been  shown  that  we  must  not  only  carefully  consider  the  amount 
of  enzymes  in  the  juice  but  also  the  zymogens.  Pawlow  and  his  pupils, 
especially  Schipowalnikoff,  have  shown  that  a  body  occurs  in  the  intes- 
tinal juice  which  activates  a  juice  otherwise  without  action  upon  proteid, 
converting  the  trypsinogen  into  trypsin.  This  body  Paw'low  calls  entero- 
kinase,  and  is  itself  without  any  solvent  action  upon  proteid.  It  is  not 
always  contained  in  the  intestinal  juice,  but  is  only  secreted  when  the 
pancreatic  juice  gets  into  the  intestine.  These  observations  were  later 
confirmed  by  others,  especially  by  Delezenne,  Camus  and  Gley,  and 
further  studied.  Enterokinase  has  also  been  found  in  all  of  the  higher  ani- 
mals examined,  and  a  kinase  with  a  similar  action  has  been  detected  by  Dele- 
zenne in  the  lymph-glands,  in  impure  fibrin,  in  bacteria  and  fungi,  and  also 
in  snake-poison.  The  enterokinase  is  made  inactive  by  heat  and  is  there- 
fore considered  as  an  enzyme.  Hamburger  and  Hekma,  who  detected 
enterokinase  in  human  intestinal  juice,  do  not  consider  it  an  enzyme  because 
a  certain  quantity  of  intestinal  juice  will  only  activate  a  certain  quantity 
of  trypsin. 

The  above  statements  concerning  the  action  of  a  varying  diet  upon  the 
enzyme  content  of  the  juice  have  been  somewhat  changed  by  the  investiga- 
tions of  Pawlow 's  school  (Lintwarew  and  others).  For  instance,  a  diet  of 
bread  and  milk  causes  the  secretion  of  a  large  quan  ity  of  juice  which  is 
rich  in  trypsinogen  but  contain;  nearly  no  trypsin.  On  giving  meat  after 
this  the  juice  also  contains  trypsin;  after  a  rich  meat  diet  the  secretion 
becomes  scant  and  the  juice  contains  only  trypsin  but  no  trypsinogen. 
The  on?  difference  between  Pawlow 's  school  and  certain  other  investi- 
gators is   as  follows:    According  to  Delezenne   and  Frouin  and  also 


1  Bernstein,  1.  c,  foot-note  4,  page  320;    Walther,  Arch,  des  sciences  biol.  de  St. 
P£tersbourg,  7. 


322  DIGESTION 

Popielski  !  the  juice  never  contains  trypsin  but  always  only  trypsinogen, 
if  it  is  collected  through  a  canula  in  Wirsung's  duct,  so  that  contact  with 
the  intestinal  mucosa  is  prevented.  Popielski  explains  the  observations 
of  Pawlow's  school  by  the  fact  that  a  contact  of  the  juice  with  the  in- 
testinal secretions  was  not  perfectly  prevented,  and  that  with  one  kind  of 
diet  a  rapid  flow  of  juice  took  place  and  with  another  a  slower  flow. 

It  is  not  clear  whether  there  are  also  kinases  for  the  other  two  en- 
Z37mes.  Pawlow's  school  claim  that  the  diastase  is  always  eliminated 
as  enzyme,  while  according  to  Pozerski  a  kinase  also  exists  for  this  zymo- 
gen. In  regard  to  steapsin  the  statements  are  somewhat  contradictory. 
According  to  Lintwarew  a  zymogen  is  secreted  with  carbohydrate  and 
fat  rich  food,  which  is  quickly  changed  into  the  enzyme  by  bile  or  intesti- 
nal juice.     With  a  meat  diet  the  steapsin  is  secreted  already  formed. 

The  specific  irritants  for  the  secretion  of  pancreatic  juice  are,  according 
to  Pawlow  and  his  collaborators,  acids  of  various  kinds,  hydrochloric  acid 
as  well  as  lactic  acid,  and  fats.  Alkalies  and  alkali  carbonates  have,  on  the 
contrary,  a  retarding  action.  It  seems  as  if  the  acids  act  in  a  reflex  man- 
ner by  irritating  the  mucosa  of  the  duodenum.  The  water,  which  causes 
a  secretion  of  acid  gastric  juice,  also  becomes  indirectly  a  stimulant  for  the 
pancreatic  secretion,  but  may  also  be  an  independent  exciter.  The  psychical 
moment  may,  at  least  in  the  first  place,  have  an  indirect  action  (secretion 
of  acid  gastric  juice),  and  the  food  seems  otherwise  to  have  an  action  on 
pancreatic  secretion  by  its  action  on  the  secretion  of  gastric  juice. 

The  most  important  excitant  for  the  secretion  of  juice  is  hydrochloric 
acid,  but  the  views  are  not  united  as  to  the  manner  in  which  the  acid  acis. 
According  to  Pawlow's  school  the  acid  acts  reflexly  upon  the  intestine,  caus- 
ing a  secretion  of  a  juice  containing  only  trypsinogen.  That  a  reflex  action  is 
here  produced  is  not  contradicted  by  the  investigations  of  Popielski. 
Wertheimer  and  Lesage,  Fleig,2  and  others.  According  to  the  researches  of 
Bayliss  and  Starling,  which  have  been  confirmed  by  Camus,  Gley,  Fleig, 
Herzen,  and  others,  a  second  factor  must  also  be  active  here.  Bay- 
liss and  Starling  have  shown  that  a  body  which  they  have  called  secretin 
ran  be  extracted  from  the  intestinal  mucosa  by  a  hydrochloric  acid  solution 
of  4  p.  m.,  and  which  when  introduced  into  the  blood  produces  a  secretion 
of  pancreatic  juice.  Secretin  is  not  destroyed  by  heat,  it  is  not  identical 
with  enterokinase,  and  is  not  considered  as  an  enzyme.  It  is  formed  from 
another  substance,  prosecretin,  by  the  action  of  acids.     We  have  numerous 

1  Delezenne  and  Frouin,  Compt.  rend.,  134,  and  Compt  rend.  soc.  biol.,  55;  Popiel- 
ski, Centralbl.  f.  Physiol.,  17,  65.  For  the  literature  on  enterokinase,  secretin  and 
pancreatic-juice  secretion  we  must  refer  to  the  extensive  literature  given  in  O.  Cohn- 
heim,  Biochem.  Centralbl.,  1,  169. 

2  Centralbl.  f.  Physiol.,  16,  681,  and  Compt.  rend.  soc.  biol.,  55.  See  also  foot- 
note, page  1. 


PANCREATIC  JUICE.  32? 

investigations  on  secretin,  but  the  statements  differ  in  several  points.  It 
is  difficult  to  obtain  a  clear  conception  of  the  amount  of  zymogens  or  en- 
zymes secreted  by  the  juice  under  the  influence  of  the  secretin.  It  seems 
to  be  clear  that  this  juice,  at  least  in  many  cases,  contains  only  trypsino- 
gen  and  no  trypsin. 

The  activation  of  the  tripsinogen  into  trypsin  may — as  the  researches 
of  Herzen,  which  have  been  substantiated  by  Gachet  and  Panchon, 
Bellamy,  Mendel  and  Rettger,  have  shown — in  life  be  brought  about  not 
only  in  the  intestine,  but  also  in  the  gland  itself.  This  activation  of  the 
trypsinogen  in  the  gland  itself  is  caused  in  a  still  unknown  manner  by  a 
body  whose  nature  is  unknown,  and  is  formed  in  the  spleen,  which  is 
congested  during  digestion.  Such  a  "charging"  of  the  pancreas  by  the 
spleen  has  been  repeatedly  suggested  by  Schiff,1  and  his  statements  have 
not  only  been  confirmed  by  these  recent  investigations,  but  in  part  also 
explained. 

The  conversion  of  the  trypsinogen  into  trypsin  in  the  removed  gland  or 
in  an  infusion  under  the  influence  of  air  and  water  and  also  by  other  bodies 
has  been  known  for  a  long  time.  According  to  Vernon  the  trypsin  itself 
has  a  strong  activating  action  upon  trypsinogen,  and  in  this  regard  it  is 
more  active  than  enterokinase.  The  ordinary  view  of  Heidenhain,  that 
the  transformation  of  trypsinogen  into  trypsin  is  also  brought  about  by 
acids,  has  been  found  to  be  incorrect  by  Hekma.2 

Another  intraglandular  enzyme  formation  in  the  pancreas  is  that  ob- 
served by  Weinland,  where  a  lactase  is  reflexly  formed  after  the  intro- 
duction of  milk-sugar  into  the  intestine.  This  is  a  special  example  of 
the  general  rule  based  upon  Brocard's3  researches,  that  the  kind  of  food 
has  a  marked  influence  upon  the  formation  of  hydrolytic  ferments  in  the 
body;  "  e'est  l'aliment  qui  fait  le  ferment." 

The  statements  as  to  the  quantity  of  pancreatic  juice  secreted  in  the 
twenty-four  hours  differ  very  much.  According  to  the  determinations 
of  Pawlow  and  his  collaborators,  Kuwschinski,  Wassiliew,  and  Ja- 
blonsky,4  the  average  quantity  (with  normally  acting  juice)  from  a  per- 
manent fistula  in  dogs  is  21.8  c.  c.  per  kilo  in  the  twenty-four  hours. 

The  pancreatic  juice  of  the  dog  is  a  clear,  colorless,  and  odorless  alka- 
line fluid  which  when  obtained  from  a  temporary  fistula  is  very  rich  in 

1  Bellamy,  Journ.  of  Physiol.,  27;  Mendel  and  Rettger,  Amer.  Journ.  of  Physiol.,  7. 
A  very  complete  reference  to  the  literature  may  be  found  in  Menia  Besbokaia:  "Du 
rapport  fonctionell  entre  le  pankr£as  et  la  rate."     Lusanne,  1901. 

:  Vernon.  Journ.  of  Physiol.,  28;  Hekma,  Kon.  Akad.  v.  Wettenschappen  te  Am- 
sterdam, 1903. 

1  Weinland,  Zeitschr.  f.  Biologie,  38  and  40;  Brocard,  Journ.  de  Physiol,  et  de 
Path.  gen.  4. 

4  Arch,  des  sciences  de  St.  P6tersbourg,  2,  391.  The  older  statements  of  Keferstein 
and  Hallwachs,  Bidder  and  Schmidt,  and  others  may  be  found  in  Kuhne,  Lehrbuch,  1 14. 


324  DIGESTION. 

proteids,  sometimes  so  rich  that  it  coagulates  like  the  white  of  the  egg 
on  heating.  Besides  proteids  the  juice  contains  also  the  three  above- 
mentioned  enzymes  (or  their  zymogens),  amylopsin,  trypsin,  steapsin,  and 
rennin,  which  was  first  observed  by  Kuhne.  Besides  the  above-mentioned 
bodies  the  pancreatic  juice  habitually  contains  small  quantities  of  leucin, 
fat,  and  soaps.  As  mineral  constituents  it  contains  chiefly  alkali  chlorides 
and  considerable  alkali  carbonate,  some  phosphoric  acid,  lime,  magnesia, 
and  iron. 

The  human  physiological  pancreatic  secretion  from  a  fistula  has  been 
recently  investigated  by  Glaessner.1  The  secretion  was  clear,  foamed 
readily,  had  a  strong  alkaline  reaction  even  towards  phenolphthalein,  and 
contained  globulin  and  albumin  but  no  proteoses  and  peptones.  The 
specific  gravity  was  1.0075  and  the  freezing-point  depression  was 
A  =-0.46-0.49°.  The  solids  were  12.44-12.71  p.  m.,  the  total  proteid 
1.28-1.74  p.  m.,  and  the  mineral  bodies  5.66-6.98  p.  m.  The  secretion 
contained  no  trypsin  but  a  proenzyme  which  was  activated  by  the  intes- 
tinal juice.  Diastase  and  lipase  were  present;  inverting  enzymes,  on  the 
contrary,  were  not.  The  daily  quantity  of  juice  was  500-800  c.  c.  The 
quantity  of  secretion,  of  ferments,  and  the  alkalinity  was  lowest  in  starvation, 
but  soon  rose  with  the  taking  of  food,  and  reached  its  maximum  in  about 
four  hours. 

The  older  analyses  of  the  juice  from  a  permanent  fistula  by  C.  Schmidt 
are  the  results  of  a  more  or  less  abnormal  secretion,  hence  we  shall  give  only 
the  analyses  of  juices  from  temporary  fistulas  on  dogs.2  The  results  are 
given  in  parts  per  1000. 

a.  b. 

Water 900.8  884.4 

Solids. 99.2  115.6 

Organic  substance 90 . 4  

Ash 8.8  

The  mineral  constituents  consisted  chiefly  of  NaCl,  7.4  p.  m. 

In  the  pancreatic  juice  of  rabbits  11-26  p.  m.  solids  have  been  found,  and  in 
that  from  sheep  14.3-36.9  p.  m.  In  the  pancreatic  juice  of  the  horse  9-15.5  p.  m. 
solids  have  been  found;  in  that  of  the  pigeon,  12-14  p.  m. 

It  has  not  been  possible  to  investigate  human  pancreatic  juice.  Never- 
theless the  fluid  obtained  from  pancreatic  cysts,  or  after  their  extirpation, 
has  been  analyzed.  As  this  fluid  cannot  give  a  perfect  idea  as  to  the 
properties  of  the  normal  juice,  we  must  refer  to  the  older  analyses  and  to 
the  recent  works  of  Schumm  and  of  Murray  and  Gies.3 

Amylopsin  or  pancreatic  diastase,  which,  according  to  Korowin  and 
Zweifel,  is  not  found  in  new-born  infants  and  does  not  appear  until  more 

1  Zeitschr.  f.  physiol.  Chem.,  40. 

2  Cited  from  Maly  in  Hermann's  Handbuch  der  Physiol.,  5,  Theil  II,  189. 

3  Schumm,  Zeitschr.  f.  physiol.  Chem.,  36;  Murray  and  Gies,  American  Medicine,  4, 
1902. 


AMYLOPSIN  AND  STEAPSIN.  325 

than  one  month  after  birth,  seems,  although  not  identical  with  ptyalin,  to 
be  nearly  related  to  it.  Amylopsin  acts  very  energetically  upon  boiled 
starch,  and  according  to  KiiiNE  upon  unboiled  starch,  especially  at  37° 
to  40°  C,  and  according  to  Vernon,1  best  at  35°  C.  It  forms,  similar  to 
the  action  of  saliva,  besides  dextrin,  chiefly  Lsomaltose  and  maltose,  with 
only  very  little  dextrose  (Museums  and  v.  Mering,  Kulz  and  Vooel  2). 
The  dextrose  is  probably  formed  by  the  action  of  the  invertin  ■  existing 
in  the  gland  and  juice.  According  to  Rachford  the  action  of  the  amylop- 
sin  is  not  reduced  by  very  small  quantities  of  hydrochloric  acid  but  is 
diminished  by  larger  amounts.  Vernon,  Grutzner  and  Wachsmann  4 
find  that  the  action  is  indeed  accelerated  by  very  small  quantities  of  hydro- 
chloric acid,  0.045  p.  m.,  while  alkalies  in  very  small  amounts  have  a  retard- 
ing action.  This  retarding  action  of  alkalies  and  hydrochloric  acid  may 
be  stopped  by  bile. 

If  the  natural  pancreatic  juice  is  not  to  be  obtained,  then  the  gland 
may  be  treated  with  water  or  glycerine.  This  infusion  or  the  glycerine  ex- 
tract diluted  with  water  (when  glycerine  has  been  used  which  has  no  reduc- 
ing action)  may  be  tested  directly  with  starch-paste.  It  is  safer,  however,  to 
first  precipitate  the  enzyme  from  the  glycerine  extract  by  alcohol,  and  wash 
with  this  liquid,  dry  the  precipitate  over  sulphuric  acid,  and  extract  witli 
water.  The  enzyme  is  dissolved  by  the  water.  The  detection  of  sugar 
may  be  performed  in  the  same  manner  as  in  the  saliva. 

Steapsin  or  Fat-splitting  Enzyme.  The  action  of  the  pancreatic  juice 
on  fats  is  twofold.  First,  the  neutral  fats  are  split  into  fatty  acids  and 
glycerine,  which  is  an  enzymotic  process;  and  secondly,  it  has  also  the 
property  of  emulsifying  the  fats. 

The  action  of  the  pancreatic  juice  in  splitting  the  fats  may  be  shown  in 
the  following  way:  Shake  olive-oil  with  caustic  soda  and  ether,  siphon  off 
the  ether  and  filter  if  necessary,  then  shake  the  ether  repeatedly  with  water 
and  evaporate  at  a  gentle  heat.  In  this  way  is  obtained  a  residue  of  fat  free 
from  fatty  acids  which  is  neutral,  and  which  dissolves  in  acid-free  alcohol 
and  is  not  colored  red  '  y  alkanet  tincture.  If  such  fat  is  mixed  with 
perfectly  fresh  alkaline  pancreatic  juice  or  with  a  freshly  prepared  infusion 
of  the  fresh  gland  and  treated  with  a  little  alkali  or  with  a  faintly  alkaline 
glycerine  extract  of  the  fresh  gland  (9  parts  glycerine  and  1  part  1  per  cent 
Eoda  solution  for  each  gram  of  the  gland),  and  some  litmus  tincture  add  d 
and  the  mixture  warmed  to  37°  C,  the  alkaline  recction  will  gradually 
disappear  and  an  acid  one  take  its  place.  This  acid  reaction  depends  upon 
the  conversion  of  the  neutral  fats  by  the  enzyme  into  glycerine  and  fr  e 
fatty  acid. 

1  Korowin,  Maly's  Jahresher.,  3;   Zweifel,  foot-note  3,  page  290;  Kiihne,  Lehrbuch, 
117;  Vernon,  Journ.  of  Physiol.,  27. 
3  See  foot-note  1,  page  291. 

8  See  Tebb,  Journ.  of  Physiol.,  15,  and  Abelous,  Compt.  rend.  Soc.  de  biol.,  43. 
*  Rachford,  Amer.  Journ.  of  Physiol.,  2;  Vernon,  1.  c. ;  Grutzner,  Pfluger's  Arch.,  91. 


326  DIGESTION. 

The  splitting  of  the  neutral  fats  may  also  be  shown  more  exactly  by 
the  following  method:  The  mixture  of  neutral  fats  (absolutely  free  from 
fatty  acids)  and  pancreatic  juice  or  pancreas  infusion  is  digested  at  the  tem- 
perature of  the  body  and  treated  with  some  soda  and  repeatedly  shaken 
with  fresh  quantities  of  ether  until  all  the  unsplit  neutral  fats  are  removed. 
Then  it  is  made  acid  with  sulphuric  acid,  and  after  the  acid  liquor  has  been 
shaken  with  ether,  the  ether  is  evaporated,  and  the  residue  tested  for  fatty 
acids. 

Another  simple  process  for  the  demonstration  of  the  fat-splitting  action 
of  the  pancreatic  glands  is  the  following  (Cl.  Bernard)  :  A  small  portion  of 
the  perfectly  fresh,  finely  divided  gland  substance  is  first  soaked  in  alcohol 
(90  per  cent).  Then  the  alcohol  is  removed  as  far  as  possible  by  pressing 
between  blotting-paper,  after  which  the  pieces  of  gland  are  covered  with  an 
ethereal  solution  of  neutral  butter-fat  (which  may  be  obtained  by  shaking 
milk  with  caustic  soda  and  ether).  After  the  evaporation  of  the  ether  the 
pieces  of  gland  covered  with  butter-fat  are  pressed  between  two  watch- 
glasses  and  then  gently  heated  to  37°  to  40°  C.  in  this  position.  After  a 
certain  time  a  marked  odor  of  butyric  acid  appears. 

The  action  of  the  pancreatic  juice  in  splitting  fats  is  a  process  analogous 
to  that  of  saponification,  the  neutral  fats  being  decomposed,  by  the  addition 
of  the  elements  of  water,  into  fatty  acids  and  glycerine  according  to  the 
following  formula:  C3H5.03.R3  (neutral  fat)  +  3H20  =  C3H5.03.H3  (glycerine) 
+  3(H.O.R)  (fatty  acid).  This  depends  upon  a  hydrolytic  splitting,  which 
was  first  positively  proved  by  Bernard  and  Berthelot.  The  pancreas- 
enzyme  also  decomposes  other  esters  just  as  it  does  the  neutral  fats  (Nencki, 
Baas).  The  fat-splitting  enzyme  of  the  pancreas  is,  according  to  Pawlow 
and  Bruno,1  aided  in  its  action  by  the  bile. 

The  fatty  acids  which  are  split  off  by  the  action  of  the  pancreatic  juice 
combine  in  the  intestine  with  the  alkalies,  forming  soaps  which  have  a 
strong  emulsifying  action  on  the  fats,  and  thus  the  pancreatic  juice  becomes 
of  great  importance  in  the  emulsification  and  the  absorption  of  the  fats. 

Trypsin.  The  action  of  the  pancreatic  juice  in  digesting  proteids  was 
first  observed  by  Bernard,  but  first  proved  by  Corvisart.2  It  depends 
upon  a  special  enzyme  called  by  Ivuhne  trypsin.  This  enzyme,  as  previ- 
ously explained,  does  not  occur  in  the  gland  as  such  but  as  trypsinogen. 
According  to  Albertoni  3  this  zymogen  is  found  in  the  gland  in  the  last 
third  of  the  intra-uterine  life.  Enzymes  more  or  less  like  trypsin  occur 
also  in  other  organs  and  are  also  very  widely  diffused  in  the  vegetable  king- 
dom,4 in  yeast,  and  in  higher  plants,  and  are  also  formed  by  various  bacteria. 

1  Bernard,  Ann.  de  chim.  et  physique  (3  se>.),  25;  Berthelot,  Jahresber.  d.  Chem., 
1855,  733;  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;  Baas,  Zeitschr.  f.  physiol.  Chem., 
14,  416;  Bruno,  Arch,  des  sciences,  biolog.  de  St.  P6tersbourg,  7,  14. 

2  Gaz.  hebdomadaire,  1857,  Nos.  15,  16,  19.  Cited  from  Bunge,  Lehrbuch,  4  Aufl., 
1  85. 

3  See  Maly's  Jahresber.,  8,  254. 

4  In  this  connection  see  Vines,  Annals  of  Botany,  16,  17,  and  Oppenheimer,  Die 
Fermente,  1900. 


TRYPSIN.  327 

Trypsin,  like  other  enzymes,  has  not  been  prepared  in  a  pure  condition. 
Nothing  is  positively  known  in  regard  to  its  nature,  but  as  obtained  thus  far  it 
shows  a  variable  behavior  (Kuhne,  Klug,  Levene,  Mays,  and  others). 
At  least  it  does  not  seem  to  be  a  nucleoproteid,  and  trypsin  has  also  been 
obtained  which  rlid  not  give  the  biuret  test  (Klug,  Mays,  Schwarzschild). 
Trypsin  dissolves  in  water  and  glycerine,  while  Kuhne  's  trypsin  was  insol- 
uble in  glycerine.  It  is  very  sensitive  to  heat,  and  even  the  body  tempera- 
ture gradually  decomposes  it  (Vernon,  Mays).  In  neutral  solution  it 
becomes  inactive  at  45°  C.  In  dilute  soda  solution  of  3-5  p.  m.  it  Is  still 
more  readily  destroyed  (BiEBNACKI,  Vernon  x).  The  presence  of  proteoses 
has,  to  a  certain  extent,  a  protective  action  on  heating  an  alkaline  trypsin 
solution.  Trypsinogen,  according  to  the  unanimous  statements  of  several 
experimenters,  is  more  resistant  towards  alkalies  than  trypsin.  Trypsin 
is  gradually  destroyed  by  gastric  juice  and  even  by  digestive  hydrochloric 
acid  alone.  Like  all  enzymes,  trypsin  is  characterized  by  its  physiological 
action,  which  consists  of  dissolving  proteid  in  alkaline,  neutral,  and  even 
in  very  faintly  acid  solutions  and  of  splitting  it  into  simpler  products, 
tuch  as  mono-  and  diamino-acids,  tryptophan,  and  other  bodies. 

The  preparation  of  pure  trypsin  has  been  tried  by  various  experimenters. 
The  most  careful  work  in  this  direction  was  done  by  Kuhne  and  Mays. 
Various  methods  have  been  suggested  by  Mays,  but  we  cannot  enter  into 
a  discussion  of  them.  A  very  pure  preparation  can  be  obtained  by  making 
use  of  the  combined  salting  out  with  NaCl  and  MgS04.  A  very  active 
solution,  and  one  that  can  be  kept  for  a  long  time  (for  more  than  twenty 
years  according  to  Hammarsten),  can  be  obtained  by  extracting  with 
glycerine  (Heidenhain  2).  An  impure  but  still  very  active  infusion  can 
be  obtained  after  a  few  days  by  allowing  the  finely  divided  gland  to  stand 
with  water  which  contains  5-10  c.  c.  chloroform  per  liter  (Salkowski)  at 
the  temperature  of  the  room.  This  infusion  can  be  kept  very  active 
for  several  years  at  the  cellar  temperature.  For  digestion  experiments 
the  active  commercial  trypsin  preparations  can  be  employed. 

The  action  of  trypsin  on  proteids  is  best  demonstrated  by  the  use  of 
fibrin.  Very  considerable  quantities  of  this  proteid  body  are  dissolved 
by  a  small  amount  of  trypsin  at  37-40°  C.  It  is  always  necessary  to  make 
&  control  test  with  fibrin  alone,  with  or  without  the  addition  of  alkali. 
Fibrin  is  dissolved  by  trypsin  without  any  putrefaction;  the  liquid  has  a 
pleasant  odor  somewhat  like  bouillon.  To  completely  exclude  putrefaction 
a  little  thymol,  chloroform,  or  toluene  should  be  added  to  the  liquid.   Tryptic 

1  Kuhne,  Verh.  d.  naturh.-med.  Vereins  zu  Heidelberg  (N.  F.),  1,  3;  Klug, 
Math,  natunv.  Ber.  aus  Ungam,  IS,  1902;  Levene,  Amer.  Journ.  of  Physiol.,  5; 
Mays,  Zeitschr.  f.  physiol.  Chem.,  38;  Vernon,  Journ.  of  Physiol.,  28  and  29;  Bier- 
nacki,  Zeitschr.  f.  Biologie,  28;  Schwarzschild,  Hofmeister's  Beitriige,  4. 

2  Pfluger's  Arch.,  10. 


328  DIGESTION. 

digestion  differs  essentially  from  pepsin  digestion  in  that  the  first  takes' 
place  in  neutral  or  alkaline  reaction  and  not,  as  is  necessary  for  peptic 
digestion,  in  an  acidity  of  1-2  p.  m.  HC1,  and  further  by  the  fact  that  the 
proteids  dissolve  in  trypsin  digestion  without  previously  swelling  up. 

As  trypsin  not  only  dissolves  proteids,  but  also  other  protein  substances 
such  as  gelatine,  this  latter  body  may  be  used  in  detecting  trypsin.  The 
liquefaction  of  strongly  disinfected  gelatine  is,  according  to  Fermi,1  a  very 
delicate  reagent  for  trypsin  or  tryptic  enzyme. 

Many  circumstances  exert  a  marked  influence  on  the  rapidity  of  the 
trypsin  digestion.  With  an  increase  in  the  quantity  of  enzyme  present  the 
digestion  is  hastened  at  least  to  a  certain  point,  and  the  same  is  also  t  ue  of 
an  increase  in  temperature  at  least  to  about  40°  C,  at  which  temperature 
the  proteid  is  very  rapidly  dissolved  by  the  trypsin.  The  reaction  is  also  of 
the  g'eatest  importance.  Trypsin  acts  energetically  in  neutral,  or  still 
better  in  alkaline,  solutions,  and  best  in  an  alkalinity  of  3-4  p.  m.  Na2C03;. 
but  the  nature  of  the  proteid  is  also  of  importance.  The  action  of  the 
alkali  depends  upon  the  number  of  hydroxyl  ions  (Dietze,  Kanitz)  and, 
according  to  Kanitz,2  the  digestion  proceeds  best  in  those  solutions, 
which  are  N/70-N/200  in  regard  to  hydroxyl  ions.  Free  mineral  acids,  even, 
in  very  small  quantities,  completely  prevent  the  digestion.  If  the  acid  is 
not  actually  free,  but  combined  with  proteid  bodies,  then  the  digestion 
may  take  place  quickly  when  the  acid  combination  is  not  in  too  great 
excess  (Chittenden  and  Cummins).  Organic  acids  act  less  disturbingly, 
and  in  the  presence  of  0.2  p.  m.  lactic  acid  and  the  simultaneous  pres- 
ence of  bile  and  common  salt  the  digestion  may  indeed  proceed  more  quickly 
than  in  a  faintly  alkaline  liquid  (Lindberger).  The  statement  of  Rach- 
ford  and  Southgate,  that  the  bile  can  prevent  the  injurious  action  of 
the  hydrochloric  acid,  and  that  a  mixture  of  pancreatic  juice,  bile,  and. 
hydrochloric  acid  digests  better  than  a  neutral  pancreatic  juice,  could 
not  be  substantiated  by  Chittenden  and  Albro.  That  bile  has  an  action 
tending  to  aid  the  tryptic  digestion  has  been  shown  by  many  investigators 
and  recently  by  Bruno,  Zuntz  and  Ussow.3 

Carbon  dioxide,  according  to  Schierbeck,4  has  a  retarding  action  in 
acid  solutions,  but  it  accelerates  the  tryptic  digestion  in  faintly  alkaline 
liquids.     Foreign  bodies,  such  as  borax  and  potassium  cyanide,  may  pro- 

1  Arch.  f.  Hygiene,  12. 

1  Kanitz,  Zeitschr.  f.  physiol.  Chem.,  37,  who  also  cites  Dietze. 

8  Chittenden  and  Cummins,  Studies  from  the  Physiol.  Chem.  Laboratory  of  Yale 
College,  New  Haven,  1885,  1,  100;  Lindberger,  Maly's  Jahresber.,  13;  Rachford  and 
Southgate,  Medical  Record,  1895;  Chittenden  and  Albro,  Amer.  Journ.  of  Physiol.,  1,. 
1898;  Rachford,  Journ.  of  Physiol.,  25;  Bruno,  1.  c;  Zuntz  and  Ussow,  Arch.  f.  (Anat. 
u.)  Physiol.,  1900. 

1  Skand.  Arch.  f.  Physiol.,  3. 


ACTION  OF  TRYPSIN.  329 

mote  tryptic  digestion,  while  other  bodies,  such  as  salts  of  mercury,  iron, 
and  others  (Chittenden  and  Cummins),  or  salicylic  acid  in  large  quan- 
tities, may  have  a  preventive  action.  The  nature  of  the  proteids  is  also 
of  importance.  Unboiled  fibrin  is,  relatively  to  most  other  proteids, 
dissolved  so  very  quickly  that  the  digestion  test  with  raw  fibrin  gives  an 
incorrect  idea  of  the  power  of  trypsin  to  dissolve  coagulated  proteid  bodies 
in  general.  Boiled  fibrin  is  digested  with  much  greater  difficulty  and 
requires  also  a  higher  alkalinity:  8  p.  m.  Na2C03  (Vernon1).  An  accumu- 
lation of  the  products  of  digestion  tends  to  hinder  the  trypsin  digestion. 

The  Products  of  the  Tr ///>//<•  Digestion.  In  the  digestion  of  unboiled 
fibrin  a  globulin  which  coagulates  at  55-60°  C.  may  be  obtained  as  an 
intermediate  product  (Herrmann  2).  Besides  this  one  obtains  from  fibrin, 
as  well  as  from  other  proteids,  the  products  previously  mentioned  in  Chapter 
II.  In  trypsin  digestion  the  cleavage  may  proceed  so  far  that  the  mix- 
ture fails  to  give  the  biuret  reaction.  This  does  not  indicate,  as  E.  Fischer 
and  Abderhalden  have  shown,  a  complete  cleavage  of  the  proteid  mole- 
cule into  mono-  and  diamino-acids,  etc.,  because  polypeptide-like  bodies 
are  produced  beside  these  acids  which  are  intermediary  bodies  between 
the  peptones  and  the  end  products.  These  bodies,  which  resist  tryptic 
digestion  for  a  long  time,  contain  the  pyrrolidin-carbonic  acid  and  phenyl- 
alanin  groups  of  the  proteids,  and  also  yield  other  monamino-acids,  such 
as  leucin,  alanin,  glutamic  acid,  and  aspartic  acid.  In  tryptic  digestion 
no  more  nitrogen  as  ammonia  is  split  eff  than  on  hydrolysis  with  acids 
(Mochiztjki),  which  is  a  difference  between  trypsin  and  the  autolytic 
enzymes.  Among  the  above-mentioned  products  we  find  on  the  auto-di"-es- 
tion  of  the  gland  other  substances,  such  as  oxyphenylethylamine  (Emerson), 
which  is  produced  from  tyrosin  by  fermentive  C02  cleavage;  also  uracil 
(Lkvene),  which  originates  from  the  nuclein  bodies,  the  purin  bases,  and 
choline,  which  latter  is  formed  from  lecithin  (Kutscher  and  Lohmann3). 
If  putrefaction  is  not  completely  prevented,  still  other  bodies  occur  which 
will  be  considered  later  in  connection  with  the  putrefactive  processes  in  the 
intestine. 

The  Action  of  Trypsin  upon  other  bodies.  The  nucleoproteids  and  nucleins 
are  so  digested  that  the  proteid  complex  is  separated  from  the  nucleic 
acid  and  then  digested.  The  nucleic  acids  may,  nevertheless,  be  somewhat, 
changed  (Araki)  ;  a  splitting,  with  the  setting  free  of  phosphoric  acid  and 
purin  bases,  does  not  seem  to  occur  with  trypsin  (Iwaxoff  4).     Gelatine  is 


1  Journ.  of  Physiol.,  28. 

2  Herrmann,  Zeitschr.  f.  physiol.  Chem.,  11. 

3  Fischer  and  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  39;  Mochizuki,  Hofmeister's 
Beitrage,  1;  Emerson,  ibid.,  1;  Levene,  Zeitschr.  f.  physiol.  Chem.,  3";  Kutscher  and 
Lohmann,  ibid.,  39. 

4Iwanoff,  Zeitschr.  f.  physiol.  Chem.,  39,  which  also  contains  the  literature. 


330  DIGESTION. 

dissolved  and  digested  by  pancreatic  juice.  A  cleavage  with  the  separa- 
tion of  glycocoll  and  leucin  does  not  occur  (Kuhne  and  Ewald),  or  only  to 
a  trivial  extent  (Reich-Herzberge  1). 

The  gelatine-forming  substance  of  the  connective  tissues  is  not  directly 
dissolved  by  trypsin,  but  only  after  it  has  been  treated  with  acids  or  soaked 
in  water  at  70°  C.  By  the  action  of  trypsin  on  hyalin  cartilage  the  cells 
dissolve,  leaving  the  nucleus.  The  matrix  is  softened  and  shows  an  indis- 
tinctly constructed  network  of  collagenous  substance  (Kuhne  and  Ewald). 
The  elastic  substance,  the  structureless  membranes,  and  the  membrane  of  the 
fat-cells,  are  also  dissolved.  Parenchymatous  organs,  such  as  the  liver  and 
the  muscles,  are  dissolved,  all  but  the  nuclei,  connective  tissue,  fat-cor- 
puscles, and  the  remainder  of  the  nervous  tissue.  If  the  muscles  are  boiled, 
then  the  connective  tissue  is  also  dissolved.  Mucin  is  dissolved  and  split 
by  trypsin,  while  chitin  and  horn  substance  do  not  seem  to  be  acted  upon 
by  the  enzyme.  Oxyhemoglobin  is  decomposed  by  trypsin  with  the  splitting 
off  of  hsematin.     Trypsin  has  no  action  upon  fat  and  carbohydrates. 

We  have  the  investigations  of  Gulewitsch,  Gonnermann,  Schwarz- 
schild,2  E.  Fischer  and  Bergell  3  upon  the  action  of  trypsin  of  simply 
constructed  substances  of  known  constitution,  such  as  acid  amides  and 
several  others  that  give  the  biuret  reaction.  An  undoubted  cleavage  of  Cur- 
tius's  biuret-base  was  first  observed  by  Schwarzschild.  He  found  that  this 
base,  which  he  considers  as  hexaglycylglycinethyl  ester,  was  decomposed 
by  trypsin  with  the  splitting  off  of  glycocoll.  Fischer  and  Bergell 
found  that  the  /^-naphthalene  sulphonic  derivatives  of  glycyl  <?-alanin  and 
<5-alanylglycin  were  very  resistant  towards  trypsin,  while  the  naphthalene 
sulpho-  and  carbethoxyl  derivatives  of  glycyl-tyrosin  were  readily  split 
by  trypsin  yielding  tyrosin.  In  the  action  of  trypsin  upon  inactive  carb- 
ethoxyl-d-1-leucin  the  asymmetric  compound  is  formed.  It  acted  especially 
upon  one-half  of  the  racemic  body  and  splits  off  1-leucin.  The  hydrolysis 
of  the  dipeptides  and  their  derivatives  is  therefore  dependent  upon  several 
factors,  namely,  upon  the  nature  of  the  amino  acids,  their  stereometric 
structure  and  other  conditions. 

Pancreatic  rennin  is  an  enzyme  found  in  the  gland  and  in  the  juice  which  coagu- 
lates neutral  or  alkaline  milk  (Kuhne  and  Roberts  and  others).  This  enzyme  is 
not  identical  with  trypsin,  and  the  optimum  of  its  action  lies  according  to  Vernon 
between  60°  and  65°.  According  to  Halliburton  and  Brodie  *  casein  is  con- 
verted by  the  pancreatic  juice  of  the  dog  into  "pancreatic  casein,"  a  sub- 
stance which,  in  regard  to  solubility,  stands  to  a  certain  extent  between  casein 
and  paracasein  (see  Chapter  XIV),  and  which  is  converted  into  paracasein  by 

1  Kuhne  and  Ewald,  Verh.  d.  naturh.-med.  Vereins  zu  Heidelberg  (N.  F.),  1;  Reich- 
Herzberge,  Zeitschr.  f.  physiol.  Chem.,  34. 

*  Hofmeister's  Beitriige,  4,  where  the  other  works  are  also  cited. 

8  Ber.  d.  d.  chem.  Gesellsch.,  36. 

♦Kuhne  and  Roberts,  Maly's  Jahrasber.,  9;  see  also  Edkins,  Journ.  of  Physiol.,  12 
(literature  references);  Halliburton  and  Brodie,  ibid.,  20;  Vernon,  ibid.,  27. 


THE  CHEMICAL  PROCESSUS  IS    THE  INTESTINE.  331 

rennin.      Further  investigations  on  the  action  of  this  enzyme   upon  milk  and 
especially  upon  pure  casein  solutions  arc  very  desirable. 

Pancreatic  calculi.  The  concrement  from  a  cystic  enlargement  of  Wirsung's 
duct  in  a  man  as  analyzed  by  Baldoni1  contained  in  1000  parts  as  follows:  Water 
34.4,  ash  120.7,  albumin  substances  34.0,  free  fatty  acids  133,  neutral  fats  124, 
cholesterin  70.9,  soaps  and  pigment  499.1,  parts. 

Besides  the  enzymes,  which  have  been  discussed  in  connection  with  the 
pancreatic  juice,  the  gland  also  contains  others,  among  which  can  be  men- 
tioned the  enzyme  which,  according  to  Stoklasa  and  his  collaborators, 
occurs  chiefly  in  organs  and  tissues  and  which  decomposes  sugar  into  alcohol 
and  carbon  dioxide,  like  zymase.  According  to  Simacek,2  in  the  pancreas 
the  glycolysis  by  means  of  alcoholic  fermentation,  and  the  hydrolysis  of 
the  disaccharides,  are  united  together  as  a  specific  action,  and  he  has 
obtained  precipitates  from  cell-free  press-fluid  with  alcohol  and  ether 
which  brought  on  both  actions  without  bacterial  action.  In  this  connec- 
tion attention  must  also  be  called  to  the  fact  that  O.  Cohnheim  3  has  been 
able  to  obtain  a  strongly  glycolytic  cell-free  fluid,  not  from  the  pancreas 
alone,  but  from  a  mixture  of  muscle  and  pancreas. 

V.    The  Chemical  Processes  in  the  Intestine. 

The  action  which  belongs  to  each  digestive  secretion  may  be  essen- 
tially changed  under  certain  conditions  by  being  mixed  with  other  digestive 
fluids  for  various  reasons,  and  also  by  the  action  of  the  enzymes  upon  each 
other;  *  and  since  the  digestive  fluids  which  flow  into  the  intestine  are 
mixed  with  still  another  fluid,  the  bile,  it  will  be  readily  understood  that 
the  combined  action  of  all  these  fluids  in  the  intestine  makes  the  chemical 
processes  going  on  therein  very  complicated. 

As  the  acid  of  the  gastric  juice  acts  destructively  on  ptyalin,  this  enzyme 
has  no  further  diastatic  action,  even  after  the  acid  of  the  gastric  juice  has 
been  neutralized  in  the  intestine.  The  bile  has,  at  least  in  certain  animals, 
a  slight  diastatic  action,  which  in  itself  can  hardly  be  of  any  great  impor- 
tance, but  which  shows  that  the  bile  has  not  a  preventive  but  rather  a 
beneficial  influence  on  the  energetic  diastatic  action  of  the  pancreatic 
juice.  Martin  and  Williams,  Pawlow  and  Bruno  5  have  observed  a 
beneficial  action  of  the  bile  on  the  diastatic  action  of  the  pancreas  infusion. 
To  this  may  be  added  that  the  organized  ferments  which  occur  habitually 
in  the  intestine  and  sometimes  in  the  food  have  partly  a  diastatic  action 
and   partly   produce   a    lactic-acid   and   butyric-acid   fermentation.     The 

1  Maly's  Jahresber.,  29,  353. 

2  Stoklasa,  see  foot-note  4,  page  9;  Simacek,  Centralbl.  f.  Physiol.,  17. 
s  Zeitschr.  f.  physiol.  Chem.,  39. 

*  See  Wroblewski  and  collaborators,  Hofmeister's  Beitrage,  1. 

6  Martin  and  Williams,  Proceed,  of  Roy.  Soc,  45  and  48;  Bruno,  foot-note  1, 
page  326. 


332  DIGESTION. 

maltose,  which  is  formed  from  the  starch,  seems  to  be  converted  into  dextrose 
in  the  intestine.  Cane-sugar  is  inverted  in  the  intestine,  and,  at  least 
in  certain  animals,  also  lactose.1  Cellulose  undergoes  a  fermentation  in  the 
intestine  by  the  action  of  micro-organisms,  producing  marsh-gas,  acetic 
acid,  and  butyric  acid  (Tappeiner);  still  it  is  not  known  to  what  extent 
the  cellulose  is  destroyed  in  this  way.2 

The  bile  has,  as  shown  by  Moore  and  Rockwood  3  and  then  especially 
by  Pfluger,  the  property  to  a  high  degree  of  dissolving  fatty  acids,  espe- 
cially oleic  acid,  which  itself  is  a  solvent  for  other  fatty  acids,  and  hence, 
as  will  be  seen  later,  it  is  of  great  importance  in  the  absorption  of  fat.  It 
is  also  of  greater  importance  that  the  bile,  as  previously  stated,  not  only 
activates  the  steapsinogen,  but  that,  as  first  shown  by  Nencki  and  Rach- 
ford,4  it  accelerates  the  fat-splitting  action  of  the  steapsin.  The  fatty  acids 
combine  with  the  alkalies  of  the  intestinal  and  pancreatic  juices  and  the 
bile,  producing  soaps  which  are  of  great  importance  in  the  absorption  of 
the  fats. 

If  to  a  soda  solution  of  about  1-3  p.  m.  Na2C03  is  added  pure,  perfectly 
neutral  olive-oil  in  not  too  large  quantity,  a  transient  emulsion  is  obtained 
after  vigorous  shaking.  If,  on  the  contrary,  one  adds  to  the  same  quantity 
of  soda  solution  an  equal  amount  of  commercial  olive-oil  (which  always 
contains  free  fatty  acids),  the  vessel  need  only  be  turned  over  for  the  two 
liquids  to  mix  and  immediately  there  appears  a  very  finely  divided  and  per- 
manent emulsion,  making  the  liquid  appear  like  milk.  The  free  fatty  acids 
of  the  commercial  oil,  which  is  always  somewhat  rancid,  combine  with  the 
alkali  to  form  soaps  which  act  to  emulsify  the  fats  (Brucke,  Gad,  Loewen- 
thal  5) .  This  emulsifying  action  of  the  fatty  acids  split  off  by  the  pancreatic 
juice  is  undoubtedly  assisted  by  the  habitual  occurrence  of  free  fatty  acids 
in  the  food,  as  well  as  by  the  splitting  off  of  fatty  acids  from  the  neutral  fats 
in  the  stomach  (see  page  306) . 

Bile  completely  prevents  peptic  zymolysis  in  artificial  digestion,  because 
it  retards  the  swelling  up  of  the  proteids.  The  passage  of  bile  into  the 
stomach  during  digestion,  on  the  contrary,  seems,  according  to  several 
investigators,  especially  Oddi  and  Dastre,8  to  have  no  disturbing  action 

1  See  footnote  3,  page  318. 

2  On  the  digestion  of  cellulose  see  Henneberg  and  Stohmann,  Zeitschr.  f.  Biologie, 
21,  613;  v.  Knieriem,  ibid.,  67;  Hofmeister,  Arch.  f.  wiss.  u.  prakt.  Thierheilkunde, 
11;  Weiske,  Zeitschr.  f.  Biologie,  22,  373;  Tappeiner,  ibid.,  20  and  24;  and  Mallevre, 
Pfliiger's  Arch.,  19;  Omeliansky,  Arch.  d.  scienc.  biol.  de  St.  Pctersbourg,  7;  E.  Miiller, 
Pfluger 's  Arch.,  83. 

3  Proceedings  of  Roy.  Soc,  60,  and  Journ.  of  Physiol.,  21.  In  regard  to  Pfliiger's 
work  see  absorption,  page  344. 

4  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;   Rachford,  Journal  of  Physiol.,  12. 

6  Brucke,  Wien.  Sitzungsber.,  61,  Abth.  2;  Gad,  Du  Bois-Reymond's  Arch.,  1878; 
Loewenthal  ibid.,  1897. 

6Oddi,  Ref.  in  Centralbl.  f.  Physiol.,  1,  312;  Dastre,  Arch,  de  Physiol.  (5),  2,  316. 


THE  CHEMICAL  PROCESSES  IN   THE  INTESTINE.  333 

on  gastric  digestion.  Bile  has  no  solvent  action  on  proteids  in  neutral 
or  alkaline  reaction,  but  still  it  may  exert  an  influence  on  proteid  digestion 
in  the  intestine.  The  acid  contents  of  the  stomach,  containing  an  abun- 
dance of  proteids,  give  with  the  bile  a  precipitate  of  proteids  and  bile-acids. 
This  precipitate  carries  a  part  of  the  pepsin  with  it  and  for  this  reason, 
and  also  on  account  of  the  partial  or  complete  neutralization  of  the  acid 
of  the  gastric  juice  by  the  alkali  of  the  bile  and  the  pancreatic  juice,  the 
pepsin  digestion  cannot  proceed  further  in  the  intestine.  On  the  contrary, 
the  bile  does  not  disturb  the  digestion  of  proteids  by  the  pancreatic  juice 
in  the  intestine.  The  action  of  these  digestive  secretions,  as  above  stated, 
is  not  disturbed  by  the  bile,  not  even  by  the  faintly  acid  reaction  due  to 
organic  acids;  but,  on  the  contrary,  the  action  of  trypsin  is  accelerated 
by  the  bile.  In  a  dog  killed  while  digestion  is  going  on,  the  faintly  acid, 
bile-containing  material  of  the  intestine  shows  regularly  a  strong  digestive 
action  on  proteids. 

The  precipitate  formed  on  the  meeting  of  the  acid  contents  of  the 
stomach  with  the  bile  easily  redissolves  in  an  excess  of  bile  and  also  in  the 
Nad  formed  in  the  neutralization  of  the  hydrochloric  acid  of  the  gastric 
juice.  This  may  take  place  even  under  faintly  acid  reaction.  Since  in 
man  the  excretory  ducts  of  the  bile  and  the  pancreatic  juice  open  near  one 
another,  in  consequence  of  which  the  acid  contents  of  the  stomach  are 
probably  immediately  in  great  part  neutralized  by  the  bile  as  soon  as  it 
enters,  it  is  doubtful  whether  a  precipitation  of  proteids  by  the  bile  occurs 
in  the  intestine. 

Besides  the  previously  mentioned  processes  caused  by  enzymes,  there 
are  others  of  a  different  nature  going  on  in  the  intestine,  namely,  the  fer- 
mentation and  putrefaction  processes  caused  by  micro-organisms.  These 
are  less  intense  in  the  upper  parts  of  the  intestine,  but  increase  in  intensity 
towards  the  lower  part  of  the  same,  and  decrease  in  the  large  intestine 
because  of  the  consumption  of  fermentable  material  and  by  the  removal 
of  water  by  absorption.  Fermentation  processes,  but  only  very  slight 
putrefaction,  occur  in  the  small  intestine  of  man.  Macfadyen,  M. 
Xexcki,  and  N.  Sieber  '  have  investigated  a  case  of  human  anus  praeter- 
naturalis, in  which  the  fistula  occurred  at  the  lower  end  of  the  ileum,  and 
they  were  able  to  investigate  the  contents  of  the  intestine  after  it  had  been 
exposed  to  the  action  of  the  mucous  membrane  of  the  entire  small  intestine. 
The  mass  was  yellow  or  yellowish  brown,  due  to  bilirubin,  had  an  acid 
reaction  which,  on  a  mixed  but  chiefly  animal  diet,  calculated  as  acetic 
acid,  amounted  to  1  p.  m.  The  contents  were  nearly  odorless,  having  an 
empyreumatic  odor  recalling  that  of  volatile  fatty  acids,  and  only  seldom 
had  a  putrid  odor  resembling  that  i  >f  indol.  The  essential  acid  present  was 
acetic  acid,  accompanied  by  fermentation  lactic  acid  and  paralactic  acid, 

1  Arch.  f.  exp.  Path.  u.  Pharm.,  2S. 


334  DIGESTION. 

volatile  fatty  acids,  succinic  acid,  and  bile-acids.  Coagulable  proteids, 
peptone,  mucin,  dextrin,  dextrose,  and  alcohol  were  present.  Leucin  and 
tyrosin  could  not  be  detected. 

According  to  the  above-mentioned  investigators,  the  proteids  are  only 
to  a  very7  slight  extent,  if  at  all,  decomposed  by  the  microbes  in  the  small 
intestine  of  man.  The  organisms  present  in  the  small  intestine  preferably 
decompose  the  carbohydrates,  forming  ethyl  alcohol  and  the  above-men- 
tioned organic  acids. 

Further  investigations  of  Jakowsky  and  of  Ad.  Schmidt  *  led  to  the 
same  result,  namely,  that  in  man  the  putrefaction  of  the  proteids  takes 
place  in  the  large  intestine.  This  putrefaction  of  the  proteids  is  not  the 
same  as  the  pancreatic  digestion.  In  putrefaction  the  decomposition  goes 
much  further  and  a  mixture  of  products  is  obtained  which  have  become 
known  through  the  labors  of  numerous  investigators,  especially  Nencki, 
Baumann,  Brieger,  H.  and  E.  Salkowski,  and  their  pupils.  The  products 
which  are  formed  in  the  putrefaction  of  proteids  are  (in  addition  to  pro- 
teoses, peptones,  amino-acids,  and  ammonia)  indol,  skatol,  paracresol,  phenol, 
phenylpropionic  acid,  and  phenylacetic  acid,  also  paraoxyphenylacetic  acid  and 
hydroparacumaric  acid  (besides  paracresol,  produced  in  the  putrefaction  of 
tyrosin),  volatile  fatty  acids,  carbon  dioxide,  hydrogen,  marsh-gas,  methylmer- 
captan,  and  sulphuretted  hydrogen.  In  the  putrefaction  of  gelatine  neither 
tyrosin  nor  indol  is  formed,  while  glycocoll  is  produced  instead. 

Among  these  products  of  decomposition  a  few  are  of  special  interest 
because  of  their  behavior  within  the  organism  and  because  after  their 
absorption  they  pass  into  the  urine.  A  few,  such  as  the  oxyacids,  pass 
unchanged  into  the  urine.  Others,  such  as  phenols,  are  directly  trans- 
formed into  ethereal  sulphuric  acids  by  synthesis,  and  are  eliminated  as 
such  by  the  urine;  on  the  contrary,  others,  such  as  indol  and  skatol,  are 
only  converted  into  ethereal  sulphuric  acids  after  oxidation  (for  details  see 
Chapter  XY).  The  quantity  of  these  bodies  in  the  urine  varies  also  with 
the  extent  of  the  putrefactive  processes  in  the  intestine;  at  least  this  is 
true  for  the  ethereal  sulphuric  acids.  Their  quantity  increases  in  the  urine 
with  a  stronger  putrefaction,  and  the  reverse  takes  place,  namely,  a  disap- 
pearance from  the  urine,  or  a  great  reduction  in  quantity,  as  Baumann, 
Harley  and  Goodbody  2  have  shown  by  experiments  on  dogs,  when  the 
intestine  was  disinfected  by  various  agents. 

Among  the  above-mentioned  putrefactive  products  in  the  intestine  the 
two  following,  indol  and  skatol,  should  be  especially  noted. 


1  Jakowsky,  Arch,  des  scienc.  biol.  de  St.  Petersbourg,  1;   Ad.    Schmidt,  Arch.  f. 
Verdauungskr.,  4. 

2  Baumann,  Zeitschr.  f.  physiol.  Chem.,  10;    Harley  and  Goodbody,  Brit.  Med. 
Journ.,  1899. 


1ND0L  AND  SKATOL.  335 

CH 

/   \ 

Indol,     C8H7N=CeH4  CH,     and     Skatol,     or      methyl-indol, 

\      / 
NH 

C.CH, 

C9H9N  =  C6H4  ^^CH,  are  two  bodies  which  stand  in  close  relationship 

\        / 
NH 

to  the  indigo  substances  and  are  formed  in  variable  quantities  from  pro- 

teid  compounds  under  different  conditions.      Hence  they  occur  habitually 

in  the  human  intestinal  canal,  and,  after  oxidation  into  indoxyl  and  skat- 

oxyl  respectively,  pass,  at  least  partly,  into  the  urine  as  the  corresponding 

ethereal  sulphuric  acids  and  also  as  glucuronic  acids. 

These  two  bodies  have  been  prepared  synthetically  in  many  ways.  Both 
may  be  obtained  from  indigo  by  reducing  it  with  tin  and  hydrochloric  acid 
and  heating  this  reduction  product  with  zinc-dust  (Baeyer  l).  Indol  may 
be  formed  from  skatol  by  passing  it  through  a  red-hot  tube.  Indol  sus- 
pended in  water  is  in  part  oxidized  into  indigo-blue  by  ozone  (Nencki  2). 

Indol  and  skatol  crystallize  in  shining  leaves,  and  their  melting-points 
are  52°  and  95°  C.  respectively.  Indol  has  a  peculiar  excrementitious 
odor,  while  skatol  has  an  intense  fetid  odor  (skatol  obtained  from  indigo  is 
odorless).  Both  bodies  are  easily  volatilized  by  steam,  skatol  more  easily 
than  indol.  They  may  both  be  removed  from  the  watery  distillate  by 
ether.  Skatol  is  the  more  insoluble  of  the  two  in  boiling  water.  Both  are 
easily  soluble  in  alcohol  and  give  with  picric  acid  a  combination  consisting  of 
red  crystalline  needles.  If  a  mixture  of  the  two  picrates  be  distilled  with 
ammonia,  they  both  pass  over  without  decomposition;  while  if  they  are 
distilled  with  caustic  soda,  the  indol  but  not  the  skatol  is  decomposed. 
The  watery  solution  of  indol  gives  with  fuming  nitric  acid  a  red  liquid  and 
then  a  red  precipitate  of  nitroso-indol  nitrate  (Nencki).  It  is  better  first 
to  add  two  or  three  drops  of  nitric  acid  and  then  a  2  per  cent  solution  of 
potassium  nitrite,  drop  by  drop  (Salkowski  3).  Skatol  does  not  give  this 
reaction.  An  alcoholic  solution  of  indol  treated  with  hydrochloric  acid 
colors  a  pine  chip  cherry-red.  Skatol  does  not  give  this  reaction.  Indol 
gives  a  deep  reddish-violet  color  with  sodium  nitroprusside  and  alkali 
(Legal 's  reaction).  On  acidifying  with  hydrochloric  acid  or  acetic  acid 
the  color  becomes  pure  blue.  Skatol  does  not  act  the  same.  The  alkaline 
solution  is  yellow  and  becomes  violet  on  acidifying  with  acetic  acid  and 
boiling.     Skatol  dissolves  in  concentrated  hydrochloric  acid  with  a  violet 

1  Annal.  d.  Chem.  u.  Pharm.,  140,  and  Supplbd.,  7,  56;  also  Ber.  d.  deutsch.  chera. 
Gesellsch.,  1. 

2  Ber.  d.  deutsch.  chem.  Gesellsch.,  8,  727,  and  ibid.,  722  and  1517. 

3  Zeitschr.  f.  physiol.  Chem.,  8,  447. 


336  DIGESTION. 

coloration.     On  warming  skatol  with  sulphuric  acid  a  beautiful  purple-red 
coloration  is  obtained  (Ciamician  and  Magnanini1). 

For  the  detection  of  indol  and  skatol  in,  and  their  preparation  from, 
excrement  and  putrefying  mixtures,  the  main  points  of  the  usual  method 
are  as  follows:  The  mixture  is  distilled  after  acidifying  with  acetic  acid; 
the  distillate  is  then  treated  with  alkali  (to  combine  with  any  phenols 
which  may  be  present)  and  again  distilled.  From  this  second  distillate  the 
two  bodies,  after  the  addition  of  hydrochloric  acid,  are  precipitated  by 
picric  acid.  The  picrate  precipitate  is  then  distilled  with  ammonia.  The  two 
bodies  are  obtained  from  the  distillate  by  repeated  shaking  with  ether  and 
evaporation  of  the  several  ethereal  extracts.  The  residue,  containing  indol 
and  skatol,  is  dissolved  in  a  very  small  quantity  of  absolute  alcohol  and 
treated  with  8-10  vols,  of  water.  Skatol  is  precipitated,  but  not  the  indol. 
The  further  treatment  necessary  for  their  separation  and  purification  will 
be  found  in  other  works. 

The  gases  which  are  produced  by  the  decomposition  processes  are  mixed 
in  the  intestinal  tract  with  the  atmospheric  air  swallowed  with  the  saliva 
and  food,  and  as  the  gas  developed  in  the  decomposition  of  different  foods 
varies,  so  the  mixture  of  gases  after  various  foods  should  have  a  dissimilar 
composition.  This  is  found  to  be  true.  Oxygen  is  found  only  in  very  faint 
traces  in  the  intestine;  this  may  be  accounted  for  in  part  by  the  formation 
of  reducing  substances  in  the  fermentation  processes  which  combine  with 
the  oxygen,  and  partly,  perhaps  chiefly,  to  a  diffusion  of  the  oxygen  through 
the  tissues  of  the  walls  of  the  intestine.  To  show  that  these  processes  take 
place  mainly  in  the  stomach  the  reader  is  referred  to  page  308,  on  the  com- 
position of  the  gases  of  the  stomach.  Nitrogen  is  habitually  found  in  the 
intestine,  and  it  is  probably  due  chiefly  to  the  swallowed  air.  The  carbon 
dioxide  originates  partly  from  the  contents  of  the  stomach,  partly  from  the 
putrefaction  of  the  proteids,  partly  from  the  lactic-acid  and  butyric-acid 
fermentation  of  carbohydrates,  and  partly  from  the  setting  free  of  carbon 
dioxide  from  the  alkali  carbonates  of  the  pancreatic  and  intestinal  juices  by 
their  neutralization  through  the  hydrochloric  acid  of  the  gastric  juice  and 
by  organic  acids  formed  in  the  fermentation.  Hydrogen  occurs  in  largest 
quantities  after  a  milk  diet,  and  in  smallest  quantities  after  a  purely  meat 
diet.  This  gas  seems  to  be  formed  chiefly  in  the  butyric-acid  fermentation 
of  carbohydrates,  although  it  may  occur  in  large  quantities  in  the  putre- 
faction of  proteids  under  certain  circumstances.  There  is  no  doubt  that  the 
methylmerca'ptan  and  sulphuretted  hydrogen  which  occur  normally  in  the 
intestine  originate  from  the  proteids.  The  marsh-gas  undoubtedly  orig- 
inates in  the  putrefaction  of  proteids.  As  proof  of  this  Ruge  2  found  26.45 
per  cent  marsh-gas  in  the  human  intestine  after  a  meat  diet.  He  found  a 
still  greater  quantity  of  this  gas  after  a  vegetable  (leguminous)  diet;   this 

1  Ber.  d.  d.  chem.  Gesellsch.,  21,  1928. 
.2  Wien.  Sitzungsber.,  44. 


INTESTINAL  PUTREFACTION.  ■'>■'>. 

coincides  with  the  observation  that  marsh-gas  may  be  produced  by  a 
fermentation  of  carbohydrates,  but  especially  of  cellulose  (Tappeixer  '). 
Such  an  origin  of  marsh-gas,  especially  in  herbivora,  is  to  be  expected. 
A  small  part  of  the  marsh-gas  and  carbon  dioxide  may  also  depend  on 
the  decomposition  of  lecithin  (Hasebroek  2). 

Putrefaction  in  the  intestine  not  only  depends  upon  the  composition  of 
the  food,  but  also  upon  the  albuminous  secretions  and  the  bile.  Among 
the  constituents  of  bile  which  are  changed  or  decomposed  there  are  not  only 
the  pigments — the  bilirubin  yields  urobilin  and  a  brown  pigment — but  also 
the  bile-acids,  especially  taurocholic  acid.  Glycocholic  acid  is  more  stable, 
and  a  part  is  found  unchanged  in  the  excrement  of  certain  animals,  while 
taurocholic  acid  is  so  completely  decomposed  that  it  is  entirely  absent  in 
the  faeces.  In  the  foetus,  on  the  contrary,  in  whose  intestinal  tract  no  putre- 
faction processes  occur,  undecomposed  bile-acids  and  bile-pigments  are 
found  in  the  contents  of  the  intestine.  The  transformation  of  bilirubin  into 
urobilin  does  not  occur,  as  previously  stated,  in  man  in  the  small  but  in  the 
large  intestine. 

That  the  secretions  rich  in  proteids  are  destroyed  in  putrefaction  in  the 
intestine  follows  from  the  fact  that  putrefaction  may  also  continue  during 
complete  fasting.  From  the  observations  of  Muller  3  upon  Cetti  it  was 
found  that  the  elimination  of  indican  during  starvation  rapidly  decreased 
and  after  the  third  day  of  starvation  it  had  entirely  disappeared,  while  the 
phenol  elimination,  which  at  first  decreased  so  that  it  was  nearly  minimum, 
increased  again  from  the  fifth  day  of  starvation,  and  on  the  eighth  or  ninth 
day  it  was  three  to  seven  times  as  much  as  in  man  under  ordinary  circum- 
stances. In  dogs,  on  the  contrary,  the  elimination  of  indican  during 
starvation  is  considerable,  but  the  phenol  elimination  is  slight.  Among 
the  secretions  which  undergo  putrefaction  in  the  intestine,  the  pancreatic 
juice,  which  putrefies  most  readily,  takes  first  place. 

From  the  foregoing  facts  it  must  be  concluded  that  the  products  formed 
by  the  putrefaction  in  the  intestine  are  in  part  the  same  as  those  formed  in 
digestion.  The  putrefaction  may  be  of  benefit  to  the  organism  in  so  far  as 
the  formation  of  such  products  as  proteoses,  peptones,  and  perhaps  also 
certain  amino  acids  is  concerned.  The  question  has  indeed  been  asked 
(Pasteur),  is  digestion  possible  without  micro-organisms?  Xuttal  and 
Thierfelder  have  shown  that  guinea-pigs  removed  from  the  uterus  of  the 
mother  by  Caesarian  section  could  with  sterile  air  digest  well  and  assimilate 
sterile  food  (milk  or  crackers)  in  the  complete  absence  of  bacteria  in  the 
intestine,  and  grew  perfectly  normal  and  increased  in  weight.     Schot- 


1  Zeitsch.  f.  Biologie,  20  and  24. 

2  Zeitschr.  f.  physiol.  Chem.,  12. 

'  Berlin,  klin.  Wochenschr.,  1887. 


338  DIGESTION. 

telius  1  has  arrived  at  other  results  by  experiments  with  hens.  The 
animal  was  hatched  under  sterile  conditions  and  was  kept  in  sterile  rooms 
and  fed  with  sterile  food,  but  had  continuous  hunger  and  ate  abundantly, 
but  soon  died  in  about  the  same  time  as  a  starving  animal.  On  mixing 
with  the  food  at  the  proper  time  a  variety  of  bacteria  from  hen  fasces,  the 
animals  gained  weight  again  and  recovered. 

The  bacterial  action  in  the  intestinal  canal  is,  at  least  in  certain  cases, 
necessary,  and  it  acts  in  the  interest  of  the  organism.  This  action  may,  by 
the  formation  of  further  cleavage  products,  be  a  loss  of  valuable  material 
to  the  organism,  and  it  is  therefore  important  that  putrefaction  in  the 
intestine  is  kept  within  certain  limits.  If  an  animal  is  killed  while  diges- 
tion in  the  intestine  is  going  on,  the  contents  of  the  small  intestine  give 
out  a  peculiar  but  not  putrescent  odor.  Also  the  odor  of  the  contents  of 
the  large  intestine  is  far  less  offensive  than  a  putrefying  pancreas  infusion 
or  a  putrefying  mixture  rich  in  proteid.  From  this  one  may  conclude  that 
putrefaction  in  the  intestine  is  ordinarily  not  nearly  so  intense  as  outside 
of  the  organism. 

It  seems  thus  to  be  provided,  under  physiological  conditions,  that 
putrefaction  shall  not  proceed  too  far,  and  the  factors  which  here  come 
under  consideration  are  probably  of  different  kinds.  Absorption  is  un- 
doubtedly one  of  the  most  important  of  them,  and  it  has  been  proved  by 
actual  observation  that  the  putrefaction  increases,  as  a  rule,  as  the  absorp- 
tion is  checked  and  fluid  masses  accumulate  in  the  intestine.  The  character 
of  the  food  also  has  an  unmistakable  influence,  and  it  seems  as  if  a  large 
quantity  of  carbohydrates  in  the  food  acts  against  putrefaction  (Hirsch- 
ler  -).  It  has  been  shown  by  Pohl,  Biernacki,  Rovighi,  Wixterxitz, 
and  Schmttz  and  others  3  that  milk  and  kephir  have  a  specially  strong 
preventive  action  on  putrefaction.  This  action  is  not  due  to  the  casein, 
but  chiefly  to  the  lactose  and  also  in  part  to  the  lactic  acid. 

A  specially  strong  preventive  action  on  putrefaction  has  been  ascribed 
for  a  long  time  to  the  bile.  This  anti-putrid  action  does  not  exist  in  neutral 
or  faintly  alkaline  bile,  which  itself  easily  putrefies,  but  to  the  free  bile- 
acid?,  especially  taurocholic  acid  (Malt  and  Emich,  Lixdberger  4).  There 
Is  no  question  that  the  free  bile-acids  have  a  strong  preventive  action  on 
putrefaction  outside  of  the  organism,  and  it  Is  therefore  difficult  to  deny 
such  an  action  in  the  intestine.  Notwithstanding  this  the  anti-putrid 
action  of  the  bile  in  the  intestine  is  not  considered  by  certain  investigators 


1  Xuttal  and  Thierf elder,  Zeitschr.  f.  physiol.  Chem.,  21  and  22;   Schottelius,  Arch, 
f.  Hygiene,  34  and  42. 

2  Hirschler,  Zeitschr.  f.  physiol.  Chem.,  10;  Zimnitzki,  ibid.,  39  (literature). 

-  hmitz,  ibid.,  1",  401,  which  gives  references  to  the  older  literature,  and  19.     See 

-  Jkowski,  Centralbl.  f.  d.  med.  Wiss.,  1893,  467,  and  Seelig,  Virchow's  Arch.,  146 
Qitera- 

4  lialy  and  Emich,  Monatshefte  f.  Chem.,  4;  Lindberger,  foot-note  3,  page  328. 


INTESTINAL  PUTREFACTION.  339 

(Voit,  Rohmann,  Hibschleb  and  Tbrbay,  Laxdauer  and  Rosenberg1) 
as  of  the  greatest   importance. 

Biliary  fistula'  have  been  established  so  as  to  study  the  importance  of 
the  bile  in  digestion  (Schwann,  Blondlot,  Bidder  and  Schmidt,2  and 
others).  As  a  result  it  has  been  observed  that  with  fatty  foods  an  imper- 
fect absorption  of  fat  regularly  takes  place  and  the  excrements  contain, 
therefore,  an  excess  of  fat  and  have  a  light-gray  or  pale  color.  The  extent 
of  deviation  from  the  normal  after  the  operation  is  essentially  dependent 
upon  the  character  of  the  food.  If  an  animal  is  fed  on  meat  and  fat,  then 
the  quantity  of  food  must  be  considerably  increased  after  the  operation, 
otherwise  the  animal  will  become  very  thin,  and  indeed  die  with  symptoms 
of  starvation.  In  these  cases  the  excrements  have  the  odor  of  carrion,  and 
this  was  considered  a  proof  of  the  action  of  the  bile  in  checking  putrefac- 
tion. The  emaciation  and  the  increased  want  of  food  depend,  naturally, 
upon  the  imperfect  absorption  of  the  fats,  whose  high  calorific  value  is 
reduced  and  must  be  replaced  by  the  taking  up  of  larger  quantities  of  other 
nutritive  bodies.  If  the  quantity  of  proteids  and  fats  be  increased,  then 
the  latter,  which  can  be  only  very  incompletely  absorbed,  accumulate 
in  the  intestine.  This  accumulation  of  the  fats  in  the  intestine  only 
renders  the  action  of  the  digestive  juices  on  proteids  more  difficult, 
and  thus  increases  the  amount  of  putrefaction.  This  explains  the  ap- 
pearance of  fetid  faeces,  whose  pale  color  is  not  due  to  a  lack  of  bile- 
pigments,  but  to  a  surplus  of  fat  (Rohmann,  Voit).  If  the  animal  is, 
on  the  contrary,  fed  on  meat  and  carbohydrates,  it  may  remain  quite 
normal,  and  the  leading  off  of  the  bile  does  not  cause  any  increased  putre- 
faction. The  carbohydrates  may  be  uninterruptedly  absorbed  in  such 
large  quantities  that  they  replace  the  fat  of  the  food,  and  this  is  the  reason 
why  the  animal  on  such  a  diet  does  not  become  emaciated.  As  with  this 
diet  the  putrefaction  in  the  intestine  is  no  greater  than  under  normal  con- 
ditions even  though  the  bile  is  absent,  it  would  seem  that  the  bile  in  the 
intestine  exercises  no  preventive  action  on  putrefaction. 

To  this  conclusion  the  objection  may  be  made  that  the  carbohydrates, 
which  are  capable  of  checking  putrefaction,  can,  so  to  speak,  undertake 
the  anti-putrid  action  of  the  bile.  But  as  there  are  also  cases  (in  dogs 
with  biliary  fistula)  where  the  intestinal  putrefaction  is  not  increased  with 
exclusive  meat  diet,3  it  is  maintained  that  the  absence  of  bile  in  the  intes- 
tine, even  by  exclusive  carbohydrate  food,  does  not  always  cause  an  in- 
creased putrefaction. 

'Voit,  Beitr.  zur  Biologie,  Jubilaumschrift,  Stuttgart,  1882;  Rohmann,  Pfluger's 
Arch.,  29;  Hirschler  and  Terray,  Maly's  Jahresber.,  26;  Landauer,  Math.  u.  Naturw. 
Ber.  aus  Ungarn,  15;  Rosenberg,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 

'Schwann,  Miiller's  Arch.  f.  Anat.  u.  Physiol.,  1844;  Blondlot,  cited  from  Bidder 
and  Schmidt,  Verdauungssiifte,  etc.,  98. 

3  See  Hirschler  and  Terray,  1.  c. 


340  DIGESTION. 

Although  the  question  as  to  the  manner  in  which  the  putrefactive 
processes  in  the  intestine  under  physiological  conditions  are  kept  within 
certain  limits  cannot  be  answered  positively,  still  it  may  be  asserted  that 
the  acid  reaction  of  the  upper  parts  of  the  intestine,  and  the  absorption  of 
water  in  the  lower  parts,  are  important  factors. 

That  the  acid  reaction  in  the  intestine  has  a  preventive  influence  on  the 
putrefactive  processes  follows  from  the  existing  relation  between  the  degree 
of  acidity  of  the  gastric  juice  and  the  putrefaction  in  the  intestine.  After 
the  investigations  and  observations  of  Kast,  Stadelmann,  Wasbutzki, 
Biernacki  and  Mester  had  proved  that  an  increased  putrefaction  in  the 
intestine  occurred  when  the  quantity  of  hydrochloric  acid  in  the  gastric  juice 
was  diminished  or  deficient,  Schmitz  *  has  lately  shown  in  man  that  on 
the  administration  of  hydrochloric  acid,  producing  a  hyperacidity  of  the 
gastric  juice,  the  putrefaction  in  the  intestine  may  be  checked.  The  ques- 
tion arises  whether  the  reaction  in  the  small  intestine  is  always  acid  and 
whether  the  acidity  is  strong  enough  to  prevent  putrefaction.  In  this 
connection  it  must  be  recalled  that  the  contents  of  the  small  intestine  are  not 
acid  due  to  hydrochloric  acid,  but  chiefly  to  organic  acids,  acid  salts,  and 
free  carbon  dioxide.  There  are  several  statements  as  to  the  reaction  of  the 
intestinal  contents,  although  they  are  somewhat  contradictory,  by  Moore 
and  Rockwood,  Moore  and  Bergin,  Matthes  and  Marquardsen,  I.  Munk, 
Nencki  and  Zalesky,  Hemmeter.2  From  these  statements  one  can  con- 
clude that  the  reaction  may  vary  not  only  among  different  animals  but 
also  in  the  same  animals  under  different  conditions.  There  is  no  doubt 
that  the  acid  reaction  in  many  cases  is  due  to  the  presence  of  organic  acids. 
On  testing  with  various  indicators  it  has  been  shown  that  sometimes  the 
upper  part,  and  often  the  lower  parts,  are  acid  due  to  acid  salts  such  as 
NaHC03  and  free  C02,  and  finally  that  in  certain  animals  the  intestinal  con- 
tents are  alkaline  throughout.  The  question  how,  under  these  conditions, 
putrefaction  is  excluded,  cannot  be  explained.  It  is  possible,  as  Bien- 
stock  3  admits,  that  the  explanation  lies  in  an  antagonistic  bacterial  action 
and  that  the  carbohydrates,  especially  lactose,  which  retard  putrefaction, 
form  a  good  nutritive  media  for  those  bacteria  which  destroy  the  putre- 
factive producers  or  retard  their  development. 

Excrements.  It  is  evident  that  the  residue  which  remains  after  com- 
plete digestion  and  absorption  in  the  intesine  must  be  different,  both 
qualitatively  and  quantitatively,  according  to  the  variet}'  and  quantity  of 

1  Zeitschr.  f.  physiol.  Chem.,  19,  401,  which  includes  all  the  pertinent  literature. 

2  Moore  and  Rockwood,  Journ.  of  Physiol.,  21;  Moore  and  Bergin,  Amer.  Journ. 
of  Physiol.,  3;  Matthes  and  Marquardsen,  Maly's  Jahresber.,  28;  Monk,  Centralbl.  f. 
Physiol.,  10;  Xencki  and  Zalesky,  Zeitschr.  f.  physiol.  Chem.,  27;  Hemmeter,  Pfliiger's 
Arch.,  SI. 

3  Arch,  f .  Hygiene,  39. 


EXCREMENTS.  341 

the  food.  In  man  the  quantity  of  excrement  from  a  mixed  diet  Is  120-150 
grams,  with  30-37  grams  of  solids,  per  twenty-four  hours,  while  the  quantity 
from  a  vegetable  diet,  according  to  Voit,1  whs  :'>:;:>  grams,  with  75  grams 

of  solids.  With  a  strictly  meat  diet  the  excrements  are  scanty,  pitch-like, 
and  colored  nearly  black.  The  scanty  excrements  in  starvation  have  a 
similar  appearance.  A  large  quantity  of  coarse  bread  yields  a  great  amount 
of  light-colored  excrement.  If  there  is  a  large  proportion  of  fat,  it  takes  a 
lighter,  clay-like  appearance.  The  decomposition  products  of  the  bile-pig- 
ments seem  to  play  only  a  small  part  in  the  normal  color  of  the  faeces. 

The  constituents  of  the  faeces  are  of  different  kinds.  In  the  excrements 
are  found  digestible  or  absorbable  constituents  of  the  food,  such  as  muscle 
fibres,  connective  tissues,  lumps  of  casein,  grains  of  starch,  and  fat,  which 
have  not  had  sufficient  time  to  be  completely  digested  or  absorbed  in  the 
intestinal  tract.  In  addition  the  excrements  contain  indigestible  bodies, 
such  as  the  remains  of  plants,  keratin  substances,  and  others;  also  form-ele- 
ments originating  from  the  mucous  coat  and  the  glands;  constituents  of 
the  different  secretions,  such  as  mucin,  cholic  acid,  dyslysin,  and  cholesterin 
(koprosterin  or  stercorin),  purin  bases,  and  enzymes;  mineral  bodies  of 
the  food  and  the  secretions;  and,  lastly,  products  of  putrefaction  or  of  the 
digestion,  such  as  skatol,  indol,  volatile  fatty  acids,  purin  bases,  lime,  and 
magnesia  soaps.  Occasionally,  also,  parasites  of  different  kinds  occur;  and 
lastly,  the  excrements  contain  micro-organisms  of  various  species. 

That  the  mucous  membrane  of  the  intestine  by  its  secretion  and  by 
the  abundant  quantity  of  detached  epithelium  contributes  essentially  to 
the  formation  of  excrement  follows  from  the  discovery  first  made  by 
L.  Hermann  and  substantiated  by  others,2  that  a  clean,  isolated  loop  of 
intestine  collects  material  similar  to  fasces.  Human  faeces  seem  to  consist 
in  greater  part  of  intestinal  secretion  and  only  in  a  smaller  part  of  residue 
from  food  on  a  meat  or  milk  diet.  Many  foods  produce  a  large  quantity 
of  faeces  chiefly  by  causing  an  abundant  secretion.3 

The  reaction  of  the  excrements  is  very  variable,  but  in  man  with  a 
mixed  diet  it  Is  neutral  or  faintly  alkaline.  It  Is  often  acid  in  the  inner 
part,  while  the  outer  layers  in  contact  with  the  mucous  coat  have  an  alka- 
line reaction.  In  nursing  infants  it  is  habitually  acid.  The  odor  is  perhaps 
chiefly  due  to  skatol,  which  was  first  found  in  the  excrements  by  Brieger, 
and  so  named  by  him.     Indol  and  other  substances  also  take  part  in  the 

1  Zeitschr.  f.  Biologie,  25,  264. 

2  Hermann,  Pfluger's  Arch.,  46.  See  also  Ehrenthal,  ibid.,  48;  Berenstein,  ibid., 
53;  Klecki,  Centralbl.  f.  Physiol.,  7,  736,  and  F.  Voit,  Zeitschr.  f.  Biologie,  29;  v. 
Moraczewski,  Zeitschr.  f.  physiol.  Chem.,  25. 

3  In  regard  to  the  constitution  of  faeces  with  various  foods  see  Hammerl,  Kermauner, 
Moeller,  and  Prausnitz,  Zeitschr.  f.  Biologie,  35,  and  Poda,  Micko,  Prausnitz  and 
Miiller.  i'6id..39. 


342  DIGESTION. 

production  of  odor.  The  color  is  ordinarily  light  or  dark  brown,  and 
depends  above  all  upon  the  nature  of  the  food.  Medicinal  bodies  may- 
give  the  faeces  an  abnormal  color.  The  excrements  are  colored  black  by 
bismuth,  yellow  by  rhubarb,  and  green  by  calomel.  This  last-mentioned 
color  was  formerly  accounted  for  by  the  formation  of  a  little  mercury 
sulphide,  but  now  it  is  said  that  calomel  checks  the  putrefaction  and  the 
decomposition  of  the  bile-pigments,  so  that  a  part  of  the  bile-pigments 
passes  into  the  faeces  as  biliverdin.  According  to  Lesage  a  green  color 
of  the  excrements  in  children  is  caused  partly  by  biliverdin  and  partly  by 
a  pigment  produced  from  a  bacillus.  In  the  yolk-yellow  or  greenish-yellow 
excrements  of  nursing  infants  one  can  detect  bilirubin.  Neither  bilirubin 
nor  biliverdin  seems  to  exist  in  the  excrements  of  mature  persons  under 
normal  conditions.  On  the  contrary,  there  is  found  stercobilin  (Masius 
and  Vanlair),  which  is  identical  with  urobilin  (Jaffe  1).  Bilirubin  may 
occur  in  pathological  cases  in  the  faeces  of  mature  persons.  It  has  been 
observed  in  a  crystallized  state  (as  haematoidin)  in  the  faeces  of  children 
as  well  as  of  grown  persons. 

The  absence  of  bile  (acholic  faeces)  causes  the  excrements  to  have,  as 
above  stated,  a  gray  color,  due  to  large  quantities  of  fat;  this  may,  however, 
be  partly  attributed  to  the  absence  of  bile-pigments.  In  these  cases  a  large 
quantity  of  crystals  has  been  observed  which  consist  chiefly  of  magnesium 
soaps  or  sodium  soaps.  Hemorrhage  in  the  upper  parts  of  the  digestive 
tract  yields,  when  it  is  not  very  abundant,  a  dark-brown  excrement,  due 
to  haematin. 

Excretin,  so  named  by  Marcet,2  is  a  crystalline  body  occurring  in  human 
excrement,  but  which,  according  to  Hoppe-Seyler,  is  perhaps  only  impure 
cholesterin  (koprosterin  or  stercorin?).  Excretolic  acid  is  the  name  given 
by  Marcet  to  an  oily  body  with  an  excrementitious  odor. 

In  consideration  of  the  very  variable  composition  of  excrements  their 
quantitative  analyses  are  of  little  value  and  therefore  will  be  omitted.3 

Meconium  is  a  dark  brownish-green,  pitchy,  mostly  acid  mass  without 
any  strong  odor.  It  contains  greenish-colored  epithelium  cells,  cell-detritus, 
numerous  fat^globules,  and  cholesterin  plates.  The  amount  of  water 
is  720-800,  and  solids,  200-280  p.  m.  Among  the  solids  there  exists  mucin, 
bile-pigments,  and  bile-acids,  cholesterin,  fat,  soaps,  traces  of  enzymes, 
calcium  and  magnesium  phosphates.  Sugar  and  lactic  acid,  soluble 
proteid  bodies  and  peptones,  also  leucin  and  tyrosin  and  the  other  pro- 
ducts of  putrefaction  occurring  in  the   intestine,  are    absent.      Meconium 

1  See  bile  pigments,  Chapter  VIII,  and  urobilin,  Chapter  XV. 

2  Annal.  de  Chim.  et  de  phys.,  59. 

3  In  regard  to  these  analyses  as  well  as  under  abnormal  conditions  and  the  litera- 
ture, see  Ad.  Schmidt  and  J.  Strassburger,  Die  Faeces  des  Menschen,  etc.  Berlin, 
1901  and  1902. 


MECONIUM.    INTESTINAL  C0NCREMENT8.  343 

may  contain  undecomposed  taurocholic  acid,  bilirubin  and  bilivefdin,  but 
it  does  not  contain  any  stercobilin,  which  is  considered  as  proof  of  the 
non-existence  of  putrefactive  p  oci'  Bee  in  the  digestive  tract  of  the  foetus. 

In  medico-legal  cases  it  Is  sometimes  necessary  to  decide  whether  spots 
on  linen  or  other  substances  are  caused  by  meconium.  In  such  cases  the 
following  conditions  exist:  The  spot  caused  by  meconium  has  a  brown- 
ish-green color  and  can  be  easily  separated  from  the  material  because,  OH 
account  of  the  ropy  property  of  the  meconium,  it  is  difficult  to  wet  through. 
When  moistened  with  water  it  does  not  develop  any  special  odor,  but  on 
warming  with  dilute  sulphuric  acid  it  smells  somewhat  fetid.  It  forms 
with  water  a  slimy,  greenish-yellow  liquid  containing  brown  (lakes.  The 
solution  gives  with  an  excess  of  acetic  acid  an  insoluble  precipitate  of 
mucin;  on  boiling  it  does  not  coagulate.  The  filtered,  watery  extract 
responds  to  Gmeltn'b,  but  still  better  to  Huppert's  reaction  for  bile-pig- 
ments. The  liquid  precipitated  by  an  excess  of  milk  of  lime  gives  a  nearly 
colorless  filtrate,  which  after  concentration  shows  Pettenkofer's  reaction. 

The  contents  of  the  intestine  under  abnormal  conditions  are  perhaps  less  the 
subject  of  chemical  analysis  than  of  an  inspection  and  microscopical  investiga- 
tion or  bacteriological  examination.  On  this  account  the  question  as  to  the 
properties  of  the  contents  of  the  intestine  in  different  diseases  cannot  be  thor- 
oughly treated  here.1 

Appendix. 

INTESTINAL   CONCREMENTS. 

Calculi  occur  very  seldom  in  human  intestine  or  in  the  intestine  of 
carnivora,  but  they  are  quite  common  in  herbivora.  Foreign  bodies  or 
undigested  residues  of  food  may,  when  for  some  reason  or  other  they  are 
retained  in  the  intestine  for  some  time,  become  incrusted  with  salts,  espe- 
cially ammonium-magnesium  phosphate  or  magnesium  phosphate,  and 
these  salts  form  usually  the  chief  constituent  of  the  concrements.  In  man 
they  are  sometimes  oval  or  round,  yellow,  yellowish  gray,  or  brownish  gray, 
of  variable  size,  consisting  of  concentric  layers  and  containing  chiefly 
ammonium-magnesium  phosphate,  calcium  phosphate,  besides  a  small  quan- 
tity of  fat  or  pigment.  The  nucleus  ordinarily  consists  of  some  foreign 
body,  such  as  the  stone  of  a  fruit,  a  fragment  of  bone,  or  something  similar. 
In  those  countries  where  bread  made  from  oat-bran  is  an  important  food 
we  often  find  in  the  large  intestine  balls  similar  to  the  so-called  hair-balls 
(  ee  below).  Such  calculi  contain  calcium  and  magnesium  phosphate 
(about  70  per  cent),  oat-bran  (15-18  per  cent),  soaps  and  fat  (about  10 
per  cent).  Concretions  which  contain  very  much  (about  74  per  cent)  fat 
seldom  occur,  and  those  consisting  of  fibrin  clots,  sinews,  or  pieces  of  meat 
incrusted  with  phosphates  are  also  rare. 

Intestinal  calculi  often  occur  in  animals,  especially  in  horses  feci  on 
bran.     These  calculi,  which  attain  a  very  large  size,  are  hard  and  heavy 

1  See  foot-note  3,  page  342. 


344  DIGESTION. 

(as  much  as  8  kilos)  and  consist  in  great  part  of  concentric  layers  of  ammo- 
nium-magnesium phosphate.  Another  variety  of  concrements  which  occurs 
in  horses  and  cattle  consists  of  gray-colored,  often  very  large,  but  relatively 
light  stones  which  contain  plant  residues  and  earthy  phosphates.  Stones 
of  a  third  variety  are  sometimes  cylindrical,  sometimes  spherical,  smooth, 
shining,  brownish  on  the  surface,  consisting  of  matted  hairs  and  plant- 
fibres,  and  termed  hair-balls.  The  so-called  "^egagropilae,"  which  prob- 
ably originate  from  the  antilopus  rupicapra,  belong  to  this  group,  and 
are  generally  considered  as  nothing  else  than  the  hair-balls  of  cattle. 

The  so-called  oriental  bezoar-stone  belongs  also  to  the  intestinal  concre- 
ments, and  probably  originates  from  the  intestinal  tract  of  the  capra 
^gagrus  and  antilope  dorcas.  There  may  exist  two  varieties  of  bezoar- 
stones.  One  is  olive-green,  faintly  shining  and  formed  of  concentric  layers. 
On  heating  it  melts  with  the  development  of  an  aromatic  odor.  It  con- 
tains as  chief  constituent  lithofellic  acid,  C20H36O4,  which  is  related  to 
cholic  acid,  and  besides  this  a  bile-acid,  lithobilic  acid.  The  others  are 
nearly  blackish  brown  or  dark  green,  very  glossy,  consisting  of  concentric 
layers,  and  do  not  melt  on  heating.  They  contain  as  chief  constituent 
ellagic  acid,  a  derivative  of  gallic  acid,  of  the  formula  C14H608,  which, 
according  to  Graebe,1  is  the  dilactone  of  hexaoxybiphenyldicarbonic  acid 
and  which  gives  a  deep-blue  color  with  an  alcoholic  solution  of  ferric  chlo- 
ride. This  last-mentioned  bezoar-stone  originates,  to  all  appearances, 
from  the  food  of  the  animal. 

Ambergris  is  generally  considered  an  intestinal  concrement  of  the  sperm- 
whale.  Its  chief  constituent  is  ambrain,  which  is  a  non-nitrogenous  substance 
perhaps,  related  to  cholesterin.  Ambrain  is  insoluble  in  water  and  is  not  changed 
by  boiling  alkalies.     It  dissolves  in  alcohol,  ether,  and  oils. 

VI,  Absorption. 

The  problem  of  digestion  consists  in  part  in  separating  the  valuable  con- 
stituents of  the  food  from  the  useless  ones  and  dissolving  or  transforming 
them  into  forms  which  are  necessary  in  the  processes  of  absorption.  In 
discussing  the  absorption  processes  we  must  treat  of  the  form  into  which 
the  different  foods  are  transformed  before  absorption,  of  the  manner  in 
which  this  is  accomplished,  and,  lastly,  of  the  forces  which  act  in  these 
processes. 

Proteids  may  not  only  be  absorbed  from  the  intestine  as  proteoses  and 
peptones,  but  also,  as  shown  by  the  earlier  investigations  of  Brucke,  Bauer 
and  Voit,  Eichhorst,  Czerny  and  Latschenberger,  and  recently  by 
Voit  and  Friedlander,2  as  non-peptonized  proteid.     In  the  researches 

1  Ber.  d.  d.  chem.  Gesellsch.,  36. 

2  Brucke,  Wien.  Sitzungsber.,  59;  Bauer  and  Voit,  Zeitschr.  f.  Biologie,  5;  Eich- 
horst, Pfluger's  Arch.,  4;  Czerny  and  Latschenberger,  Virchow's  Arch.,  59 j  Voit  and 
Friedlander,  Zeitschr.  f.  Biologie,  33. 


ABSORPTION.  345 

of  the  two  last-mentioned  investigators  neither  casein  (as  milk)  nor  hydro- 
chloric-acid myosin,  nor  acid  albuminate  (in  acid  solution)  was  absorbed, 
while,  on  the  contrary,  about  21  per  cent  of  ovalbumin  or  seralbumin  and 
69  per  cent  of  alkali  albuminate  (dissolved  in  alkali)  were  absorbed.  The 
proteids  of  the  thymus  gland  (nucleoproteids)  are,  according  toMocHizuKi,1 
absorbed  well  after  introduction  into  the  rectum.  Under  these  conditions 
the  question  arises,  To  what  extent  are  the  proteids  absorbed  as  peptone  or 
proteoses  or  in  other  forms? 

This  question  cannot  be  decisively  answered.  The  observations  made 
by  various  investigators,  Schmidt-Mulheim  (on  dogs),  Ellenberger  and 
Hofmeister  (on  pigs),  Ewald  and  Gumlich  (on  man),  as  is  to  be  expected, 
are  contradictor}-.  A  part  of  the  digested  products  are  absorbed  in  the 
stomach,  in  which  organ,  according  to  Schmidt-Mulheim,  the  absorption 
and  digestion  run  parallel  and  the  dissolved  products  leave  the  stomach 
more  or  less  rapidly  and  pass  into  the  intestine,  where  they  are  exposed  to 
a  further  cleavage.  According  to  the  recent  investigations  of  E.  Zuxz  and 
Reach  2  chiefly  proteoses  and  little  of  the  further-removed  digestive  prod- 
ucts are  formed  in  the  stomach  of  the  dog.  These  last  products  are  more 
readily  absorbed  than  the  proteoses  and  for  this  reason  the  chief  quantity 
of  dissolved  proteids  in  the  stomach  consists  of  proteoses. 

In  what  way  are  the  proteoses  and  peptones  absorbed,  and  how  are  they 
conveyed  to  the  tissues?  The  generally  accepted  view  is  that  they  do  not 
pass  into  the  blood  through  the  lymphatics,  but  through  the  intestinal 
epithelium,  and  this  view  is  based  essentially  on  the  two  following  conditions. 
On  completely  isolating  the  chyle  from  the  blood  circulation,  the  proteid 
absorption  from  the  intestine  is  not  impaired  (Ludwig  and  Schmidt- 
Mulheim)  ;  and  on  a  diet  rich  in  proteid  the  quantity  thereof  in  the  chyle 
(in  man)  was  not  noticeably  increased  (Munk.  and  Rosexstein).  Asher 
and  Barbera  3  have  recently,  it  is  true,  shown  in  experiments  on  a  dog 
that  the  quantity  of  proteid  in  the  lymph  was  a  little  increased  after  par- 
taking of  an  abundance  of  proteid.  This  experiment  does  not  disprove  the 
assertion  of  Muxk,  that  the  blood-vessels  form  nearly  the  exclusive  exit  of 
the  proteids  from  the  intestinal  tract. 

After  a  diet  rich  in  proteids  neither  proteoses  nor  peptone  are  found 
in  the  blood  or  the  chyle.     Nor  are  they  present  in  the  urine;    and  the 


1  Maly's  Jahresber.,  31,  517.  In  regard  to  the  absorption  of  gelatine  in  the  intes- 
tine see  Reach,  Pfluger's  Arch.,  86. 

2  Schmidt-Mulheim,  Du  Bois-Reymond 's  Arch.,  1879;  Ellenberger  and  Hofmeister, 
ibid.,  1890;  Ewald  and  Gumlich,  Berlin,  klin.  Wochenschr. ,  1890;  E.  Zunz,  Hof- 
meister's  Beithige,  3;  Reach,  ibid.,  5. 

3  Schmidt-Mulheim,  Du  Bois-Reymond 's  Arch.,  1877;  Munk  and  Rosenstein,  Vir- 
chow's  Arch.,  123;  Asher  and  Barbara,  Centralbl.  f.  Physiol.,  11,  403;  Munk,  ibid., 
11,  585.     See  also  Mendel,  Amer.  Journ.  Physiol.,  2. 


346  DIGESTION. 

absence  of  these  bodies  in  the  blood  after  digestion  cannot  be  explained  by 
the  statement  that  they,  like  the  proteoses  (peptone)  injected  subcutane- 
ously  or  directly  into  the  blood,  are  quickly  eliminated  through  the  kidneys 
(Plosz  and  Gyergyai,  Hofmeister,  Schmidt-Mulheim1).  It  might  be 
supposed  that  the  proteoses  (peptone)  formed  in  digestion  are  retained  by 
the  liver,  and  that  this  is  the  reason  why  they  are  not  found  in  the  blood. 
This  explanation  does  not  seem  to  be  sufficient.  Neumeister  has  inves- 
tigated the  portal  blood  of  rabbits  in  whose  stomachs  large  quantities  of 
proteoses  and  peptone  had  been  introduced,  without  finding  traces  of  the 
body  in  question.  He  has  also  shown  that  when  the  liver  of  a  dog  is  sup- 
plied with  the  portal-blood  to  which  peptone  is  added  (ampho-peptone),  this 
is  not  retained  by  the  liver.  Shore  has  arrived  at  similar  results  in  regard 
to  the  importance  of  the  liver,  and  has  also  shown  that  the  spleen  cannot 
transform  peptone.  Peptone  seems  to  pass  neither  into  the  blood  nor  the 
chylous  vessels,  and  the  following  observation  of  Ludwig  and  Salvioli  2 
bears  out  this  assumption.  These  investigators  introduced  a  peptone  solu- 
tion into  a  double-ligatured,  isolated  piece  of  the  small  intestine,  which 
was  kept  alive  by  passing  defibrinated  blood  through  it,  and  observed  that 
the  peptone  disappeared  from  the  intestine,  but  that  the  blood  passing 
through  did  not  contain  any  peptone. 

It  must  be  remarked  in  connection  with  this  view  that,  according  to 
Embden  and  Knoop,3  proteoses  sometimes  occur  in  the  blood  of  dogs, 
although  thus  far  no  connection  has  been  detected  between  their  occurrenee 
and  the  absorption  of  proteid  in  the  intestine.  This  occurrence  of  pro- 
teoses in  the  blood  is  not  contradictory  to  the  view  that  the  chief  quantity 
of  proteoses  does  not  pass  from  the  intestine  into  the  blood  as  such. 

Many  observations  indicate  that  the  proteoses  and  peptone  are  trans- 
formed in  some  way  in  the  intestine  or  intestinal  wall,  and  a  retransforma- 
tion  of  proteoses  into  proteid  is  considered  as  the  most  plausible. 

Certain  investigators,  such  as  v.  Ott,  Nadine  Popofp,  and  Julia 
Brinck:,4  are  of  the  opinion  that  the  proteoses  and  peptone  of  gastric 
digestion  are  transformed  into  seralbumin  before  they  pass  into  the  walls  of 
the  digestive  tract.  This  transformation  is  brought  about  by  means  of  the 
epithelium  cells,  as  also  by  the  living  activity  of  a  fungus  called  by  Julia 
Brinck  micrococcus  restituens.  No  positive  proofs  have  been  presented  to 
support  this  view. 

1  Plosz  and  Gyergyai,  Pfliiger's  Arch.,  10;  Hofmeister,  Zeitschr.  f.  physiol.  Chem., 
5;  Schmidt-Mulheim,  Du  Bois-Reymond's  Arch.,  1880. 

2  Neumeister,  Sitzungsber.  d.  phys.-med.  Gesellsch.  zu  Wurzburg,  1889,  and  Zeitschr. 
f.  Biologie,  24;  Shore,  Journ.  of  Physiol.,  11;  Salvioli,  Du  Bois-Reymond's  Arch.,  1880, 
Suppl. 

3  Hofmeister 's  Beitrage,  3. 

4  v.  Ott,  Du  Bois-Reymond's  Arch.,  1883;  Popoff,  Zeitschr.  f.  Biologie,  25;  Brinck, 
ibid.,  453. 


ABSORPTION.  347 

The  view  that  the  transformation  of  the  proteoses  and  peptone  takes 
place  after  they  have  been  taken  up  by  the  mucous  membrane  has  better 
foundation.  The  observations  of  Hofmeister,1  according  to  whom  the 
walls  of  the  stomach  and  the  intestine  are  the  only  parts  of  the  body  in 
which  proteoses  (peptone)  occur  constantly  during  digestion,  and  also 
that  proteoses  (peptone)  at  the  temperature  of  the  body  after  a  time  dis- 
appeared from  the  excised  but  apparently  still  living  mucous  coat  of  the 
stomach,  confirm  this. 

This  disappearance  of  proteoses  is  considered  by  Hofmeister  as  a 
transformation  into  ordinary  proteid.  For  such  a  transformation  of  pro- 
teoses in  the  mucosa  of  the  stomach,  Glaessner  2  has  suggested  new  ex- 
perimental evidence,  while  the  Hofmeister  school  (Embden  and  Knoop) 
consider  the  regeneration  of  peptone  into  coagulable  proteid  in  the  intes- 
tine as  not  proven. 

According  to  Hofmeister  the  leucocytes,  which  are  increased  during 
digestion,  play  an  important  part  in  the  transformation  of  the  proteoses 
and  peptones.  They  may  take  up  the  proteoses  (peptone)  and  be  the 
means  of  transporting  them  to  the  blood,  and  secondly  by  their  growth, 
regeneration,  and  increase  may  stand  in  close  relationship  to  the  trans- 
formation and  assimilation  of  the  bodies.  Heidexhaix,  who  considers  that 
the  transformation  of  peptone  into  proteid  in  the  mucous  membrane  is 
positively  settled,  does  not  attribute  so  great  an  importance  to  the  leuco- 
cytes in  the  absorption  of  the  peptones,  chiefly  on  the  ground  of  compara- 
tive estimation  of  the  quantity  of  absorbed  peptones  and  leucocytes.  He 
considers  it  as  more  probable  that  the  reconversion  of  the  peptones  into 
proteid  takes  place  in  the  epithelium  layers.  This  view  is  further  corrobo- 
rated by  the  investigations  of  Shore.3 

On  account  of  the  discovery  of  erepsin  by  Cohxheim  the  theory  as  to 
the  absorption  of  proteids  has  taken  another  direction.  There  seems  to 
be  a  tendency  to  lean  towards  the  view  that  the  proteoses  and  peptones 
are  split  in  the  intestine,  or  in  the  intestinal  mucosa,  into  simpler  bodies 
which  do  not  give  the  biuret  test  and  from  which  the  proteids  are  regen- 
erated. The  question  whether  the  active  agents  are  erepsin  (Cohxheim) 
or  trypsin  (Seemaxx  and  Ktjtscheh)  is  only  of  secondary  importance,  as 
both  of  these  enzymes  split  the  proteoses  and  peptones  alike.  According 
to  Embdex  and  Kxoop  *  the  intestinal  wall  when  perfectly  free  from  trypsin 
cannot  cause  the  disappearance  of  bodies  giving  the  biuret  test. 

According  to  the  investigations  of  the  Hofmeister  school  on  pepsin  diges- 

1  Zeitschr.  f.  physiol.  Chem.,  6,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  19,  20,  and  22. 
'  Hofmeister 's  Beitriige,  1. 
6  Heidenhain,  Pfliiger's  Arch.,  43;  Shore,  1.  c. 

4  O.  Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  33,  35,  36;  Kutscher  and  Seemann,  ibid., 
34,  35;  Embden  and  Knoop,  1.  c. 


34S  DIGESTION. 

tion,  and  of  Fischer  and  Abderhalden  on  trypsin  digestion  (see  Chapter  II), 
the  disappearance  of  the  biuret  test  does  not  indicate  a  cleavage  of  the 
proteids  into  amino-acids,  since  peptoids  or  polypeptides  occur;  consequently 
it  is  for  the  present  not  possible  to  say  to  what  extent  the  proteids  are 
broken  down  in  the  intestinal  canal,  and  how  far  the  proteids  are  absorbed 
as  proteoses  or  peptones  or  as  simpler  products.  Although  there  are  the 
feeding  experiments  of  Loewi  1  with  the  products  of  the  auto-digestion  of 
the  pancreas,  one  cannot  say  anything  definite  as  to  the  supposed  proteid 
synthesis  from  the  absorbable  products  (those  not  giving  the  biuret  test)  of 
the  proteid  cleavage  in  the  intestinal  canal.  It  must  not  be  forgotten  that 
according  to  Nencki  and  Zaleski  2  large  amounts  of  ammonia  are  formed 
from  the  cleavage  products  of  the  proteids  in  the  intestinal  tract  during 
digestion. 

The  extent  of  the  proteid  absorption  is  dependent  essentially  upon  the 
kind  of  food  introduced,  since  as  a  rule  the  protein  substances  from  an 
animal  source  are  much  more  completely  absorbed  than  from  a  vegetable 
source.  As  proof  of  this  the  following  observations  are  given:  In  his  experi- 
ments on  the  utilization  of  certain  foods  in  the  intestinal  canal  of  man  Rub- 
ner  found  that  with  an  altogether  animal  diet,  on  partaking  of  an  average 
of  738-884  grams  of  fried  meat  or  948  grams  of  eggs  per  day,  the  nitrogen 
deficit  with  the  excrement  was  only  2.5-2.8  per  cent  of  the  total  introduced 
nitrogen.  With  a  strict  milk  diet  the  results  were  somewhat  unfavorable, 
since  after  partaking  of  4100  grams  of  milk  the  nitrogen  deficit  increased 
to  12  per  cent.  The  conditions  are  quite  different  with  vegetable  food,  as 
shown  by  the  experiments  of  Meyer,  Rubner,  Hultgren  and  Lander- 
gren,  who  made  experiments  with  various  kinds  of  rye  bread  and  found  that 
the  loss  of  nitrogen  through  the  faeces  amounted  to  22-48  per  cent.  Ex- 
periments with  other  vegetable  foods,  and  also  the  investigations  of  Schus- 
ter, Cramer,  Meinert,  Mori,3  and  others  on  the  utilization  of  foods  with 
mixed  diets,  have  led  to  similar  results.  With  the  exception  of  rice,  wheat 
bread,  and  certain  very  finely  divided  vegetable  foods,  it  is  found  in  general 
that  the  nitrogen  deficit  by  the  faeces  increases  with  a  larger  quantity  of 
vegetable  material  in  the  food. 

The  reason  for  this  is  manifold.  The  large  quantity  of  cellulose  frequently 
present  in  vegetable  foods  impedes  the  absorption  of  proteids.  The  greater 
irritation  produced  by  the  vegetable  food  itself  or  by  the  organic  acids 

1  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  48.  See  also  Henderson  and  Dean,  Amer. 
Journ.  of  Physiol.,  9. 

2  Arch.  d.  scienc.  biol.  de  St.  Pdtersbourg,  4;  Salaskin,  Zeitschr.  f.  physiol.  Chem., 
25;  Nencki  and  Zaleski,  Arch.  f.  exp.  Path.  u.  Pharm.,  37. 

3  Rubner,  Zeitschr.  f.  Biologie,  15;  Meyer,  ibid.,  7;  Hultgren  and  Landergren, 
Nord.  mod.  Arch.,  21;  Schuster,  in  Voit's  "Untersuch.  d.  Kost,"  etc.,  142;  Cramer, 
Zeitschr.  f.  physiol.  Chem.,  G;  Meinert,  " Ueber  Massennahrung, "  Berlin,  1885;  Kell- 
ner  and  Mori,  Zeitschr.  f.  Biologie,  25. 


ABSORPTION.  349 

formed  in  the  fermentation  in  the  intestinal  canal  causes  a  more  violent 
peristalsis,  which  drives  the  contents  of  the  intestine  faster  than  otherwise 
along  the  intestinal  canal.  Another  and  most  important  reason  is  the 
fact  that  a  part  of  the  vegetable  protein  substances  seems  to  be  indi- 
gestible. 

In  speaking  of  the  functions  of  the  stomach  we  stated  that  after  the 
removal  or  excision  of  this  organ  an  abundant  digestion  and  absorption  of 
prut,  ids  may  take  place.  It  is  therefore  of  interest  to  learn  how  the  diges- 
tion and  absorption  of  proteids  go  on  after  the  extirpation  of  the  second 
proteid-digesting  organ,  the  pancreas.  In  this  connection  there  are  the 
observations  on  animals  after  complete  or  partial  extirpation  of  the  gland 
by  Minkowski  and  Abelmaxx,  Sand.meyer,  v.  Harley,  after  destroying 
the  gland  by  Rosenberg,  and  also  in  man  after  closing  the  pancreatic  duct 
by  Harley,  Deucher.1  In  all  these  different  cases  such  discrepant  figures 
have  been  obtained  for  the  utilization  of  the  proteids — between  80  per 
cent  after  the  apparently  complete  exclusion  of  pancreatic  juice  in  man 
(Deucher)  and  18  per  cent  after  extirpation  of  the  gland  in  dogs 
(Harley) — that  one  can  hardly  draw  any  clear  conception  as  to  the  extent 
and  importance  of  the  trypsin  digestion  in  the  intestine. 

The  carbohydrates  are,  it  seems,  chiefly  absorbed  as  monosaccharides. 
Dextrose,  lsevulose,  and  galactose  are  probably  absorbed  as  such.  The  two 
disaccharides,  saccharose  and  maltose,  ordinarily  undergo  an  inversion  in 
the  intestinal  tract  and  are  converted  into  dextrose  and  laevulose.  Lactose 
is  also,  at  least  in  certain  animals,  inverted  in  the  intestine.  In  other 
mature  animals,  on  the  contrary,  if  the  lactase  formation  is  not  excited  by 
milk  food,  it  is  not  inverted  or  only  to  a  slight  extent  (Voit  and  Lusk, 
Weinland,  Portier,  Rohmann  and  Xagano),  and  it  probably  is  absorbed 
as  such  in  these  animals  if  it  does  not  undergo  fermentation,  or,  as  Roh- 
maxx  and  Nagano  2  considered,  if  it  is  not  transformed  in  the  intestinal 
mucosa  in  some  unknown  way.  An  absorption  of  non-inverted  carbo- 
hydrates is  not  improbable,  and  according  to  Otto  and  v.  Mkring  3  the 
portal  blood  contains  besides  dextrose  a  dextrin-like  carbohydrate  after 
a  carbohydrate  diet.  A  part  of  the  carbohydrates  is  destroyed  by  fermen- 
tation in  the  intestine,  with  the  formation  of  lactic  and  acetic  acids  and 
other  bodies. 

The  different  varieties  of  sugars  are  absorbed  with  varying  degrees  of 

1  Abelmann,  "Ueber  die  Ausniitzung  der  Nahrungsstoffe  nach  Pankreasexstirpa- 
tion"  (Inaug.-Dissert.  Dorpat,  1890),  cited  from  Maly's  Jahresber.,  20;  Sandmeyer, 
Zeitschr.  f.  Biologie,  31;  Rosenberg,  Pfliiger's  Arch.,  70;  Harley,  Journ.  of  Pathol, 
and  Bacteriol.,  1S95;  Deucher,  Correspond.  Blatt.  f.  Schweiz.  Aerzte,  2S. 
.  2  Voit  and  Lusk,  Zeitschr.  f.  Biologie,  2S;  Rohmann  and  Xagano,  Pfliiger's  Arch., 
95,  which  contains  the  references  to  the  literature. 

3  Otto,  see  Maly's  Jahresber.,  17;  v.  Mering,  Du  Bois-Reymond 's  Arch.,  1877. 


350  DIGESTION. 

rapidity,  but  as  a  general  thing  absorption  occurs  very  quickly.  This 
absorption  takes  place  quicker  in  the  upper  part  of  the  intestine  than  the 
lower  part  (Rohmann,  Lannois  and  Lupine,  Rohmann  and  Nagano  l). 
It  is  generally  admitted  that  the  simpler  sugars  are  more  quickly  split 
than  the  disaccharides,  while  the  statements  as  to  the  absorption  of  the 
clisaccharides  differ  somewhat  (Hedon,  Albertoni,  Hober,  Waymouth 
Reid,  Rohmann  and  Nagano).  There  seems  to  be  no  doubt  but  that 
lactose  is  absorbed  slower  than  the  two  other  disaccharides.  According  to 
the  extensive  experiments  of  Rohmann  and  Nagano  saccharose  is  ab- 
sorbed quicker  than  maltose.  Nagano  2  contends  that  the  pentoses  are 
absorbed  slower  than  the  hexoses. 

On  the  introduction  of  starch  even  in  very  considerable  quantities  into 
the  intestinal  tract  no  dextrose  passes  into  the  urine,  which  probably  de- 
pends in  this  case  upon  the  absorption  and  assimilation  and  the  slow  sac- 
charification  taking  place  simultaneously.  If,  on  the  contrary,  large  quantities 
of  sugar  are  introduced  at  one  time,  then  an  elimination  of  sugar  by  the 
urine  takes  place,  and  this  elimination  of  sugar  is  called  alimentary  glyco- 
suria. In  these  cases  the  assimilation  of  the  sugar  and  the  absorption  do 
not  occur  at  the  same  time,  hence  the  liver  and  the  remaining  organs  do 
not  have  the  necessary  time  to  fix  and  utilize  the  sugar.  This  glycosuria 
may  also  in  part  be  due  to  the  fact  that  the  introduction  of  considerable 
quantities  of  sugar  forces  this  body  to  be  absorbed  not  only  in  the  ordinary 
way  through  the  blood-vessels  to  the  liver  (see  below),  but  also  in  part  by 
passing  into  the  blood  circulation  through  the  lymphatic  vessels,  thus  evad- 
ing the  liver. 

That  quantity  of  sugar  to  which  we  must  raise  the  ingested  substance  in 
order  to  produce  an  alimentary  glycosuria  gives,  according  to  Hofmeister,3 
the  assimilation  limit  for  that  same  sugar.  This  limit  is  different  for  various 
kinds  of  sugar;  and  it  also  varies  for  the  same  sugar  not  only  in  different 
animals,  but  also  for  different  members  of  the  same  species,  as  also  for  the 
same  individual  under  different  circumstances.  In  general  it  can  be  said 
that  in  regard  to  the  ordinary  varieties  of  sugar,  such  as  dextrose,  lsevulose, 
saccharose,  maltose,  and  lactose,  the  assimilation  limit  is  highest  for  dex- 
trose and  lowest  for  lactose.  It  must  be  admitted  that  with  an  overabundant 
quantity  of  sugars  in  the  intestinal  tract  the  disaccharides  do  not  have 
sufficient  time  for  their  complete  inversion,  and  this  has  been  directly  shown 
by  Roilmaxx  and  Nagano.  It  is,  therefore,  not  remarkable  that  also 
disaccharides  have  been  found  in  the  urine  in  cases  of  alimentary  glycosuria.4 

1  Lannois  et  L6pine,  Arch,  de  Physiol.  (3),  1;  Rohmann,  Pfliiger's  Arch.,  41;  see 
also  foot-note  2,  page  349. 

2  In  regard  to  the  literature  on  the  absorption  of  sugars  see  foot-note  2,  page  349. 

3  Arch.  f.  exp.  Path.  u.  Pharm.,  2">  and  20. 

4  For  the  literature  in  regard  to  the  passage  of  various  kinds  of  sugars  into  the  urine 


ABSORPTION.  351 

The  investigations  of  Ludwig  and  v.  M eking  and  others  have  explained 
how  the  sugars  enter  into  the  blood-stream,  namely,  that  they  as  well 
as  bodies  soluble  in  water  do  not  ordinarily  pass  over  into  the  chylous 
vessels  in  measurable  quantities,  but  arc  in  greatest  part  taken  up  by  the 
blood  in  the  capillaries  of  the  villi  and  in  this  way  pass  into  the  mass  of 
the  blood.      These  investigations  have  been  confirmed  by  observations  of 

1.  MuNK  and  RoSENSTEIN  '  on  human  beings. 

The  reason  why  the  sugars  and  other  soluble  bodies  do  not  pass  over 
into  the  chylous  vessels  in  appreciable  quantity  is,  according  to  Heiden- 
hain,2  to  be  found  in  the  anatomical  conditions,  in  the  arrangement  of  the 
capillaries  close  under  the  layer  of  epithelium.  Ordinarily  these  capillaries 
find  the  necessary  time  for  the  removal  of  the  water  and  the  solids  dis- 
solved in  it.  But  when  a  large  quantity  of  liquid,  such  as  a  sugar  solution, 
is  introduced  into  the  intestine  at  once,  this  is  not  possible,  and  in  these 
cases  a  part  of  the  dissolved  bodies  passes  into  the  chylous  vessels  and  the 
thoracic  duct  (Ginsberg  and  Rohmann3). 

The  introduction  of  larger  quantities  of  sugar  into  the  intestine  at  one 
time  can  readily  cause  a  disturbance  with  diarrhceal  evacuations  of  the 
intestine.  If  the  carbohydrate  is  introduced  in  the  form  of  starch,  then 
very  large  quantities  may  be  absorbed  without  causing  any  disturbance, 
and  the  absorption  may  be  very  complete.  Rubner  found  the  following: 
On  partaking  508-670  grams  of  carbohydrates,  as  wheat  bread,  per  day 
the  part  not  absorbed  amounted  to  only  0.8-2.6  per  cent.  For  peas,  where 
357-588  grams  were  eaten,  the  loss  was  3.6-7  per  cent,  and  for  potatoes 
(718  grams)  7.6  per  cent.  Constantinidi  found  on  partaking  367-380 
grams  of  carbohydrates,  chiefly  as  potatoes,  a  loss  of  only  0.4-0.7  per  cent. 
In  the  experiments  of  Rubner,  as  also  of  Hultgren  and  Landergren,4 
with  rye  bread  the  utilization  of  carbohydrates  was  less  complete,  although 
the  loss  in  a  few  cases  rose  even  to  10.4-10.9  per  cent.  It  at  least  follows 
from  the  experiments  made  thus  far  that  man  can  absorb  more  than  500 
grams  of  carbohydrates  per  diem  without  difficulty. 

We  generally  consider  the  pancreas  as  the  most  important  organ  in  the 
digestion  and  absorption  of  amylaceous  bodies,  and  it  is  a  question  how 
these  bodies  are  absorbed  after  the  extirpation  of  the  pancreas.  As  on  the 
absorption  of  proteids,  so  also  on  the  absorption  of  starch,  the  observations 
have  given  variable  results.     In  certain  cases  the  absorption  was  nearly 

see  C.  Voit,  Ueber  die  Glykogenbildung,  Zeitschr.  f.  Biologie,  28,  and  F.  Voit,  foot-note 

2,  page  250.    See  also  Blumenthal,  Zur  Lehre  von  der  Assimilationsgrenze  der  Zucker- 
arten,  Inaug.-Dissert.  1903,  Strassburg. 

1  v.  Mering,  Du  Bois- Raymond's  Arch.,  1877;  Munk  and  Rosenstein,  1.  c. 

2  Pftiiger's  Arch.,  43,  Suppl, 

'Ginsberg,  Pfliiger's  Arch.,  44;   Rohmann,  ibid.,  41. 

4  Rubner,  Zeitschr.  f.  Biologie,  15  and  19;  Constantinidi,  ibid.,  23;  Hultgren  and 
Landergren,  1.  c. 


352  DIGESTION. 

nil,  while  in  others  it  was,  on  the  contrary,  rather  impaired,  and  with 
dogs  devoid  of  pancreas  it  has  been  found  that  of  the  starch  partaken  the 
absorption  was  decreased  50  per  cent  (Rosenberg,  Cavazzani1). 

Emulsification  seems  to  be  of  the  greatest  importance  in  the  absorption 
of  fats,  and  this  emulsion  occurs  in  the  chyle  on  the  introduction  into  the 
intestine  of  not  only  neutral  fats,  but  also  of  fatty  acids.  The  fatty  acids 
do  not  exist  as  such  in  the  emulsified  fat  of  the  chyle.  The  investigations 
of  I.  Munk,  later  confirmed  by  others,  have  shown  that  the  fatty  acids 
undergo  in  great  part  a  synthesis  into  neutral  fats  in  the  walls  of  the  intes- 
tine, and  carried  as  such  by  the  stream  of  chyle  into  the  blood.  This 
synthesis  seems  to  take  place  in  the  mucous  membrane  (Moore  2). 

The  assumption  that  the  fat  is  absorbed  chiefly  as  an  emulsion  is  partly 
based  on  the  abimdance  of  emulsified  fat  in  the  chyle  after  feeding  with  fat, 
and  partly  on  the  fact  that  a  fat  emulsion  is  often  found  in  the  intestine 
after  such  food.  As  an  abundant  cleavage  of  neutral  fats  occurs  in  the 
intestinal  canal,  and  also  as  the  fatty  acids  do  not  occur  in  the  chyle  as 
such,  but  as  emulsified  fat  after  a  synthesis  with  glycerine  into  neutral  fats, 
it  is  to  be  doubted  whether  the  emulsified  fat  of  the  chyle  originates  from 
an  absorption  of  emulsified  fat  in  the  intestine  or  from  a  subsequent  emul- 
sification of  neutral  fats  formed  synthetically.  This  doubt  has  greater 
warrant  in  that  Frank  3  has  shown  that  the  fatty-acid  ethyl  ester  is  abun- 
dantly taken  up  by  the  chyle  from  the  intestine,  not  as  such,  but  as  split-off 
fatty  acids  from  which  then  the  neutral  emulsified  fats  of  the  chyle  are 
formed. 

The  assumption  of  an  absorption  of  fats  as  an  emulsion  contradicts  the 
fact  that  an  emulsion  produced  by  means  of  soaps  is  not  permanent  in  an 
acid  liquid;  hence  we  cannot  consider  the  presence  of  an  emulsion  in  the 
intestine  as  possible  as  long  as  it  is  acid.  This  difficulty  is  not  too  serious, 
as  the  reaction  is  often  due  to  only  carbonic  acid  and  bicarbonates  and 
also  as  found  by  Kuhne  and  recently  shown  by  Moore  and  Krumbholz,4 
the  proteids  have  a  preserving  action  upon  fat  emulsions.  The  older  views 
as  to  fat  absorption  were  that  the  fat  was  absorbed  as  soaps,  soluble  in 
water,  as  well  as  finely  emulsified  fat,  and  this  last  form  was  considered  as 
of  the  greatest  importance.  This  view  has  recently  undergone  essential 
modifications,  due  to  the  work  of  Moore  and  Rockwood,  and  especially 
to  the  extensive  work  of  Pfluger.5 

1  Cavazzani,  Centralbl.  f.  Physiol.,  7.     See  also  foot-note  1,  page  349. 

2  Munk,  Virchow's  Arch.,  80.  See  also  v.  Walther,  Du  Bois-Reymond's  Arch., 
1890;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21;  Frank,  Zeitschr.  f.  Biologie,  36; 
Moore,  see  Biochem.  Centralbl.,  1,  741. 

3  Zeitschr.  f.  Biologie,  36. 

*  Kuhne,  Lehrb.  der  physiol.  Chem.,  122;  Moore  and  Krumbholz,  Journ.  of  Physiol., 
22. 

5  In  regard  to  the  newer  literature  on  fat  absorption  we  can  refer  to  the  works  of 


ABSORPTION.  353 

Moore  and  Rockwood  have  shown  the  great  solvent  action  of  the  bile 
for  fatty  acids,  and  on  continuing  these  investigations  further,  Moore  and 
Parker  have  found  that  the  bile  increases  the  solubility  of  soaps  in  water 
and  can  prevent  their  gelatinization,  a  fact  which  is  of  greater  importance 
for  the  absorption  of  fats  than  the  solubility  of  the  fatty  acids  in  bile.  The 
extent  of  lecithin  in  the  bile  is  of  great  importance  for  the  solubility  therein 
of  the  fatty  acids  as  well  as  the  soaps.  According  to  the  above-mentioned 
investigators  the  absorption  of  fat  from  the  intestine  is  essentially  dependent 
upon  the  solubility  of  the  soaps  and  free  fatty  acids  in  the  bile.  The  neutral 
fats  are  split  and  the  free  fatty  acids  are  in  part  absorbed,  dissolved  as  such 
by  the  bile,  and  in  part  combined  with  alkalies,  forming  soaps.  Neutral 
fats  are  regenerated  from  the  fatty  acids,  and  the  alkali  set  free  from  the 
soaps  is  secreted  back  again  into  the  intestine  and  used  for  the  re-formation 
of  soaps. 

The  importance  of  the  bile,  the  soaps,  and  the  alkali  carbonates  has  been 
closely  studied,  chiefly  by  very  thorough  investigations  of  Pfluger.  He 
has  quantitatively  determined  the  solvent  power  of  the  above-mentioned 
bodies — alone  as  well  as  different  mixtures  of  these — for  the  various  fatty 
acids,  and  has  closely  studied  the  mode  of  action  of  the  bile.  From  his 
investigations  he  has  arrived  at  the  conclusion  that  no  unsplit  fat  is  absorbed, 
that  all  fats,  before  their  absorption,  must  first  be  split  into  glycerine  and 
fatty  acids,  and  that  the  bile,  on  account  of  its  solvent  power  for  soaps 
and  fatty  acids,  is  sufficient  for  the  absorption  of  large  quantities  of  fat 
eaten.  The  object  of  the  formation  of  an  emulsion  is,  according  to  this  view, 
that  the  fat  in  this  condition  forms  such  a  large  surface  for  the  action  of 
the  steapsin  or  the  fat-splitting  agents. 

The  possibility  that  all  the  fat  must  be  first  split  and  that  no  unsplit 
fat  is  absorbed  is,  according  to  these  researches,  not  to  be  denied.  It  is 
the  opinion  of  the  author  that  it  is  still  too  early  to  give  a  positive  verdict 
as  to  how  these  conditions  in  the  intestine  are  brought  about  and  the  con- 
clusion must  be  left  for  further  investigations. 

The  next  question  is  whether  all  the  fat  or  the  greater  part  of  the  same 
passes  into  the  blood  through  the  lymphatics  and  the  thoracic  duct.  Accord- 
ing to  the  researches  of  Walther  and  Frank  x  on  dogs,  it  seems  that  only 
a  small  part  of  the  fats,  or  at  least  of  the  fatty  acids  fed  passes  into  the 
chylous  vessels;  but  these  observations  can  hardly  be  applied  to  the  absorp- 
tion of  neutral  fats,  or  to  the  absorption  in  man  under  normal  circumstances. 
Mr  xk  and  Rosenstein  2  in  their  investigations  on  a  girl  with  a  lymph  fistula 
found  60  per  cent  of  the  fat  ingested  in  the  chyle,  and  of  the  total  quan- 

Pfliiger,  Pfluger 's  Arch.,  80,  81,  82,  85,  88,  89,  and  90,  where  the  work  of  other  investi- 
gators is  cited  and  discussed. 

1  Walther,  Du  Bois-Reymond's  Arch.,  1900;  Frank,  ibid.,  1892. 

'Virchow's  Arch.,  123. 


354  DIGESTION. 

tity  of  fat  in  the  chyle  only  4-5  per  cent  existed  as  soaps.  On  feeding  with 
a  foreign  fatty  acid,  such  as  erucic  acid,  they  found  37  per  cent  of  the  intro- 
duced body  as  neutral  fat  in  the  chyle. 

The  completeness  with  which  fats  are  absorbed  depends,  under  normal 
conditions,  essentially  upon  the  land  of  fat.  In  this  regard  it  is  known, 
especially  from  the  investigations  of  Mtjnk  and  Arnschink,1  that  the 
varieties  of  fat  with  high  melting-points,  such  as  mutton-tallow  and  espe- 
cially stearin,  are  not  so  completely  absorbed  as  the  fats  with  low  melting- 
points,  such  as  hog-  and  goose-fat,  olive-oil,  etc.  The  kind  of  fat  also  has 
an  influence  upon  the  rapidity  of  absorption,  as  Mtjnk  and  Rosenstein 
found  that  solid  mutton-fat  was  absorbed  more  slowly  than  fluid  lipanin. 
The  extent  of  absorption  in  the  intestinal  tract  is,  under  physiological  con- 
ditions, very  considerable,  In  the  case  of  a  dog  investigated  by  Voit  it 
was  found  that  out  of  350  grams  of  fat  (butter)  partaken,  346  grams  were 
absorbed  from  the  intestinal  canal,  and  according  to  the  investigations  of 
Rubner  2  the  human  intestine  can  absorb  over  300  grams  of  fat  per  diem. 
The  fats  are,  according  to  Rubner,  much  more  completely  absorbed  when 
free,  in  the  form  of  butter  or  lard,  than  when  enclosed  in  the  cell-membranes, 
as  in  bacon. 

Claude  Bernard  showed  long  ago  with  experiments  on  rabbits  in  which 
the  ductus  choledochus  was  made  to  open  into  the  small  intestine  above  the 
pancreatic  duct,  that  after  food  rich  in  fats  the  chylous  vessels  of  the  intes- 
tine above  the  pancreas  passages  were  transparent,  while  below  they  were 
milk-white,  and  also  that  the  bile  alone  cannot  produce  an  absorption  of 
the  emulsified  fat  without  the  pancreatic  juice.  Dastre  3  has  performed 
the  reverse  experiment  on  dogs.  He  tied  the  ductus  choledochus  and 
adjusted  a  biliary  fistula  so  that  the  bile  flowed  into  the  intestine  below 
the  mouth  of  the  pancreatic  passages.  On  killing  the  animal  after  a  meal 
rich  in  fat  the  chylous  vessels  were  first  found  milk-white  below  the  dis- 
charge of  the  biliary  fistula.  From  this  Dastre  draws  the  conclusion  that 
a  combined  action  of  the  bile  and  pancreatic  juice  is  important  in  the 
absorption  of  fats — a  conclusion  which  stands  in  good  accord  with  the 
experience  of  many  others. 

Through  numerous  observations  of  many  investigators,  such  as  Bidder 
and  Schmidt,  Voit,  Rohmann,  Fr.  Muller,  I.  Munk,4  and  others,  it  has 
been  shown  that  the  exclusion  of  the  bile  from  the  intestinal  tract  diminishes 
the  absorption  of  fat  to  such  an  extent  that  only  one  seventh  to  about  one 
half  of  the  quantity  of  fat  ordinarily  absorbed  undergoes  absorption.     In 


1  Munk,  Virchow's  Arch.,  80  and  95;   Arnschink,  Zeitschr.  f.  Biolgie,  26. 

2  Voit,  Zeitschr.  f.  Biologic,  9;   Rubner,  ibid.,  15. 

3  Arch,  de  Physiol.  (5),  2. 

*  F.  Muller,  Sitzungsber.  de  phys.-raed.  Gesellsch.  zu  Wiirzburg,  1885;    I.  Munk, 
Virchow's  Arch.,  122.     See  also  foot-notes  1  and  2,  page  339. 


ABSORPTION.  355 

icterus  with  entire  exclusion  of  the  bile  a  considerable  decrease  in  the 
absoqition  of  fat  is  noticed.  As  under  normal  conditions,  so  also  in  the 
absence  of  bile  in  the  intestine,  the  more  readily  melting  parts  of  the  fat 
are  more  completely  absorbed  than  those  which  have  a  high  melting-point. 
1.  Mr\K  found  in  his  experiments  on  dogs  with  lard  and  mutton-tallow 
that  the  absorption  of  the  high-melting  tallow  was  reduced  twice  as  much 
as  the  lard  on  the  exclusion  of  the  bile  from  the  intestine. 

We  also  loam  from  the  investigations  of  Rohmann  and  I.  Munk  that 
in  the  absence  of  bile  the  relationship  between  fatty  acids  and  neutral  fats 
is  changed,  namely,  about  80-90  per  cent  of  the  fat  existing  in  the  faeces 
consists  of  fatty  acid,  while  under  normal  conditions  the  faeces  contain 
1  part  neutral  fat  to  about  2-2£  parts  free  fatty  acids.  It  is  not  possible 
ate  how  this  increased  quantity  of  fatty  acids  in  the  fat  of  the  faeces 
is  produced  upon  the  exclusion  of  the  bile  from  the  intestine. 

There  is  no  doubt  that  the  bile  is  of  great  importance  in  the  absorption 
of  fats.  Still  there  is  also  no  doubt  that  rather  considerable  quantities  of 
fat  may  be  absorbed  from  the  intestine  in  the  absence  of  bile.  What  rela- 
tion does  the  pancreatic  juice  bear  to  this  question? 

Tpon  this  point  a  rather  large  number  of  observations  on  animals  have 
been  made  by  Abelmann  and  Minkowski,  Sandmeyer,  Harley,  Rosen- 
berg, Hedon  and  Ville,  and  also  on  man  by  Fr.  Muller  and  Deucher.1 
In  all  of  these  investigations  a  more  or  less  diminished  absorption  of  fat  was 
observed  after  the  extirpation  or  destruction  of  the  gland,  or  the  exclu- 
sion of  the  juice  from  the  intestine.  The  results  are  very  diverse  as  to 
the  extent  of  this  diminution,  as  in  certain  cases  no  absorption  of  fat  was 
observed,  while,  on  the  contrary,  a  considerable  absorption  was  noted  in 
the  same  class  of  animal  (dog)  and  even  in  the  same  animal.  According 
to  Minkowski  and  Abelmann,  after  the  total  extirpation  of  the  pancreas 
the  fat  of  the  food  introduced  is  not  absorbed  at  all,  with  the  exception  of 
milk,  of  which  28-53  per  cent  of  its  fat  is  absorbed.  Other  investigators 
have  obtained  other  results,  and  Harley  has  observed  a  case  where  in  a 
dog  an  absorption  of  only  4  per  cent  of  the  milk-fat,  or,  on  the  complete 
exclusion  of  intestinal  bacteria,  even  no  absorption,  took  place.  The  con- 
ditions may  be  somewhat  different  in  the  different  cases;  but  it  is  certain 
that  the  absence  of  pancreatic  juice  from  the  intestine  essentially  affects 
the  fat  absorption.  It  is  also  just  as  certain  that  the  absorption  of  fat  is 
most  abundant  in  the  simultaneous  presence  of  bile  as  well  as  pancreatic 
juice  in  the  intestine.     A  little  fat  may  still  be  absorbed  even  in  the  absence 


1  Muller,  "Unter.  liber  den  Icterus,"  Zeitschr.  f.  klin.  Med.,  12;  H£don  and  Ville, 
Arch,  de  Physiol.  (5),  9;  Harley,  Journ.  of  Physiol.,  18,  Journ.  of  Pathol,  and  Bacteriol., 
1895,  and  Proceed.  Roy.  Soc,  61.  In  regard  to  the  other  authors  see  foot-note  1, 
page  349. 


356  DIGESTION. 

of  these  two  fluids,  as  shown  by  the  investigations  of  Hedon  and  Ville 
and  Cunningham.1 

The  reason  for  the  fact  that  the  fat  absorption  is  diminished  in  the 
absence  of  bile  from  the  intestine  must  be  sought  for  in  the  above-mentioned 
role  of  this  fluid.  It  is  more  difficult  to  state  why  the  absence  of  pan- 
creatic juice  causes  a  reduction  in  the  absorption  of  fat.  The  most  natu- 
ral view  is  that  the  neutral  fats  are  here  less  completely  split,  but  this 
does  not  seem  to  be  the  case  because  the  non-absorbed  fat  of  the  faeces 
consists,  on  the  exclusion  of  bile  and  pancreatic  juice  (Minkowski  and 
Abelmann,  Harley,  Hedon  and  Ville,  Deucher),  chiefly  of  free  fatty 
acids.  A  still  unknown  change  caused  by  gastric  lipase  or  by  micro- 
organisms or  otherwise  may  produce  a  cleavage  of  the  fat  in  these  cases. 
The  imperfect  fat  absorption  after  the  extirpation  of  the  pancreas  can 
possibly  be  explained  by  the  removal  of  a  considerable  part  of  the  alkalies 
necessary  for  the  formation  of  the  emulsion  and  for  the  solution  of  the 
fatty  acids,  but  as  Sandmeyer  found  in  dogs  deprived  of  their  pancreas 
that  the  fat  absorption  was  raised  by  giving  chopped  pancreas  with  the 
fat,  this  can  hardly  be  a  sufficient  explanation. 

The  soluble  salts  are  also  absorbed  with  the  water.  The  proteids, 
which  can  dissolve  a  considerable  quantity  of  salts,  such  as  earthy  phos- 
phates which  are  otherwise  insoluble  in  alkaline  water,  are  of  great  im- 
portance in  the  absorption  of  such  salts. 

The  soluble  constituents  of  the  digestive  secretions  may,  like  other  dis- 
solved bodies,  be  absorbed,  as  is  demonstrated  by  the  passage  of  pepsin 
into  urine;  the  enzymes  may  also  be  absorbed.  The  occurrence  of  uro- 
bilin in  urine  attests  the  absorption  of  the  bile-constituents  under  physio- 
logical conditions  despite  the  fact  that  the  occurrence  of  very  small  traces 
of  bile-acids  in  the  urine  is  disputed.  The  absorption  of  bile-acids  by  the 
intestine  seems  to  be  positively  proved  by  other  observations.  Tap- 
peiner  2  introduced  a  solution  of  bile-salts  of  a  known  concentration  into 
an  intestinal  knot  and  after  a  time  investigated  the  contents.  He  found 
that  in  the  jejunum  and  the  ileum,  but  not  in  the  duodenum,  an  absorption 
of  bile-acids  took  place,  and  further  that  of  the  two  bile-acids  only  the 
glycocholic  acid  was  absorbed  in  the  jejunum.  Further,  Schiff  long  ago 
expressed  the  opinion  that  bile  undergoes  an  intermediate  circulation,  in 
such  wise  that  it  Is  absorbed  from  the  intestine,  then  carried  to  the 
liver  by  the  blood,  and  lastly  eliminated  from  the  blood  by  this  organ. 
Although  this  view  has  met  with  some  opposition,  still  its  correctness  seems 
to  be  established  by  the  researches  of  various  investigators,  and  more 
recently  by  Prevost  and  Binet,  and  specially  by  Stadelmann  and  his 


1  H6don  and  Ville,  1  ,c. ;  Cunningham,  Journ.  of  Physiol.,  23. 

2  Wien.  Sitzungsber.,  77. 


ABSORPTION.  357 

pupils.1  After  the  introduction  of  foreign  bile  into  the  intestine  of  an 
animal  the  foreign  bile-acids  appear  again  in  the  secreted  bile. 

How  does  the  removal  of  large  portions  of  the  various  parts  of  the 
intestine  affect  absorption?  Harley  2  has  been  able  to  perform  a  partial 
extirpation  of  the  large  intestine  and  in  another  instance  a  complete  extir- 
pation. This  last  condition  increased  the  faeces  considerably,  especially 
because  of  the  large  increase  in  the  water  (fivefold).  Fats  and  carbohy- 
drates were  absorbed  just  as  completely  as  in  the  normal.  The  absorp- 
tion of  the  proteids,  on  the  contrary,  was  reduced  to  only  84  per  cent  as 
compared  to  93-98  per  cent  in  normal  dogs.  After  extirpation  the  faeces 
sometimes  did  not  contain  any  urobilin  or  only  traces  thereof,  while  bile- 
pigments  existed  in  large  amounts. 

Erlanger  and  Hewlett  3  found  that  dogs,  where  70-83  per  cent  of 
the  total  length  of  the  jejunum  and  ileum  had  been  removed,  could  be 
kept  alive  like  other  animals  if  only  the  food  was  not  too  rich  in  fat.  When 
the  food  contained  large  amounts  of  fat  then  25  per  cent  was  evacuated  by 
the  faeces  as  compared  to  4-5  per  cent  in  the  normal  animal.  Under  these 
same  conditions  the  amount  of  nitrogen  in  the  faeces  was  increased  to 
twice  the  normal  amount. 

After  the  exclusion  of  the  colon  in  rabbits  Bergmann  and  Hultgren  4 
could  not  determine  any  action  upon  the  availability  of  the  cellulose  and 
also  no  diminution  in  the  utility  of  the  other  constituents  of  the  food  could 
be  observed. 

The  question  as  to  the  forces  which  are  active  in  the  intestine  during 
absorption  has  not  been  answered.  It  is  certain  that  thus  far  the  laws  of 
diffusion  and  osmosis  alone  are  not  sufficient  to  explain  absorption  although 
the  views  are  disputed.  With  all  these  facts  in  view  and  as  it  is  not 
within  the  scope  of  this  book  to  enter  more  in  detail  upon  the  numerous 
investigations  on  this  subject,  we  must  refer  to  larger  works  5  and  to  text- 
books on  physiology  for  further  information. 

1  Schiff,  Pfliiger's  Arch.,  3;  Prevost  and  Binet,  Compt.  rend.,  106;  Stadelmann, 
see  foot-note  2,  page  261. 

2  Proceed.  Roy.  Soc,  64. 

'  Araer.  Journ.  of  Physiol.,  6. 
<Skand.  Arch.  f.  Physiol.,  14. 

5  See  Hober,  Physikalische  Chemie  der  Zelle,  Leipzig,  1902,  and  I.  Munk,  Ergeb- 
nisse  der  Physiologie,  I,  Abt.  1. 


CHAPTER  X. 
TISSUES   OF  THE  CONNECTIVE   SUBSTANCE. 

I.  The  Connective  Tissues. 

The  form-elements  of  the  tj'pical  connective  tissues  are  cells  of  various 
kinds,  of  a  not  very  well  known  chemical  composition,  and  gelatine-yielding 
fibrils,  which,  like  the  cells,  are  imbedded  in  an  interstitial  or  intercellular 
substance.  The  fibrils  consist  of  collagen.  The  interstitial  substance  con- 
tains chiefly  mucoid  (tendon-mucoid) ,  besides  serglobulin  and  seralbumin, 
which  occur  in  the  parenchymatous  fluid  (Loebisch  *). 

The  connective  tissue  also  often  contains  fibres  or  formations  consisting 
of  elastin,  sometimes  in  such  great  quantities  that  the  connective  tissue 
is  transformed  into  elastic  tissue.  A  third  variety  of  fibres,  the  reticular 
fibres,  also  occur,  and  according  to  Siegfried  these  consist  of  reticulin. 

If  finely  divided  tendons  are  extracted  in  cold  water  or  NaCl  solutions,  the 
proteid  bodies  soluble  in  the  nutritive  fluid  in  addition  to  a  little  mucoid  are 
dissolved.  If  the  residue  is  extracted  with  half-saturated  lime-water,  then 
the  mucoid  is  dissolved  and  may  be  precipitated  from  the  filtered  extract 
by  saturating  with  acetic  acid.  The  extracted  residue  contains  the  fibrils 
of  the  connective  tissue  together  with  the  cells  and  the  elastic  substance. 

The  so-called  tendon  mucin  is  not  true  mucin,  but  a  mucoid,  which, 
as  first  shown  by  Levene  and  then  by  Cutter  and  Gies,  contains  a  part  of 
its  sulphur  as  an  acid  related  to  chondroitin-sulphuric  acid.y^  These  mucoids, 
which  according  to  Cutter  and  Gies  are  mixtures  of  several  glucoproteids, 
contain  2.2-2.33  per  cent  sulphur,  as  shown  by  the  analyses  of  Chittenden 
and  Gies,  as  well  as  those  of  Cutter  and  Gies.  The  quantity  of  sulphur 
split  off  as  sulphuric  acid  was  1.33-1.62  per  cent  (Cutter  and  Gies  2).  .. 
""""  "~"h"e*fibrils  of  the  connective  tissue  are  elastic  and  swell  slightly  in  water, 
somewhat  more  in  dilute  alkalies  or  in  acetic  acid.  On  the  other  hand, 
they  shrink  by  the  action  of  certain  metallic  salts,  such  as  ferrous  sulphate 
or  mercuric  chloride,  and  tannic  acid,  which  form  insoluble  combinations 
with  the  collagen.  Among  these  combinations,  which  prevent  putrefaction 
of  the  collagen,  that  with  tannic  acid  has  been  found  of  the  greatest  tech- 

1  Zeitschr.  f.  physio  I.  Chem.,  10. 

2  Levene,  ibid.,  31  and  39;  Cutter  and  Gies,  Amer.  Journ.  of  Physiol,  6;  Chittenden 
and  Gies,  Maly's  Jahresber.,  26. 

358 


CONNECT!}  E   TISSUES.  359 

nical  importance  in  the  preparation  of  leathery  In  regard  to  the  collagens, 
gelatines,  elastins,  and  reticulins,  sec  pages  59  04. 

The  tissues  described  under  the  names  mucous  or  gelatinous  tissues  are 
characterized  more  by  their  physical  than  their  chemical  properties  ami  have 
been  bu1  little  studied.  So  much,  however,  is  known,  thai  the  mucous  or 
gelatinous  tissues  contain,  at  least  in  certain  cases,  as  in  the  acaleplue.  no 
mucin. 

The  umbilical  cord  is  the  most  accessible  material  for  the  investigation 
of  the  chemical  constituents  of  the  gelatinous  tissues.  The  mucin  occurring 
therein  has  been  described  on  page  52.  C.  Th.  Morner  1  has  found  a 
mucoid  in  the  vitreous  humor  which  contains  12.27  per  cent  nitrogen  and 
1 .1 '.»  per  cent  sulphur. 

Young  connective  tissue  is  richer  in  mucoid  than  old.  Halliburton2 
found  an  average  of  7.66  p.  m.  mucoid  in  the  skin  of  very  young  children 
and  only  3.85  p.  m.  in  the  skin  of  adults.  In  so-called  myxcedema,  in 
which  a  reformation  of  the  connective  tissue  of  the  skin  takes  place,  the 
quantity  of  mucoid,  is  also  increased. 

-^The  connective  tissue  and  also  the  elastic  tissue  are  richer  in  water  and 
poorer  in  solids  in  young  animals  as  compared  to  full-grown  animals.  This 
may  be  seen  from  the  following  analyses  of  the  achilles  tendon  (Buerger 
and  Gies),  and  of  the  ligamentum  nuchae  (Vaxdegrift  and  Gies  3).      ^/ 

Achilles  tendon.  Ligament. 


Calf.                          Ox.                             Calf.  Ox. 

Water 675 . 1  p.  m.  628 . 7  p.  m.  651 . 0  p.  m.  575  7  p  m 

Solids 324.9    "  371.3    "  394.0    "  124  3    " 

( >Tganic  bodies 318.4    "  366.6    "  342.4    "  419.6    " 

Inorganic  bodies 6.1"             4.7"                 6.6"  J  7" 

Fat 10.4     "  11.2     " 

Proteid 2.2    "  6.16" 

Mucoid 12 .  83  "  5  25  " 

Elastin 16.33"  316  70" 

Collagen 315.88"  72.30" 

Extractives,  etc 8.96"  7.99" 

In  regard  to  the  mineral  bodies  it  must  be  remarked  that  according  to 
the  determinations  of  H.  Schulz  *  the  connective  tissue  is  rich  in  silicic 
acid.  The  greatest  amount  was  found  by  him  in  the  crystalline  lens  of 
the  ox,  namely,  0.5814  gram  per  kilo  of  dried  substance.  In  man  he  found 
0.0637  gram  in  the  tendons,  0.1064  gram  in  the  fascia,  and  0.244  gram  in 
Wharton's  jelly  for  every  kilo  of  dried  substance.  The  quantity  of  silicic 
acid  is  higher  in  the  young  than  in  the  old;   in  man  it  is  highest  in  the 

1  Zeitschr.  f.  physiol.  Chem.,  IS,  2.10. 

-  Mucin  in  Myxcedema.  Further  Analyses.  Kings  College.  Collected  Papers  No.  1, 
1893. 

s  Buerger  and  Gies.  Amer.  Journ.  of  Physiol.,  6;  Vandegrift  and  Gies,  ibid.,  5. 
1  Pfluger's  Arch.,  84  and  89. 


360  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

embryonic  connective  tissue  of  the  umbilical  cord.  In  the  last  Schulz 
found  also  0.403  gram  Fe203,  0.693  gram  MgO,  3.297  grams  CaO,  and  3.794 
grams  P205  for  every  kilo  of  dried  substance. 

II.  Cartilage. 

■^  Cartilaginous  tissue  consists  of  cells  and  an  original  hyaline  matrix, 
which,  however,  may  become  changed  in  such  wise  that  there  appears  in  it 
a  network  of  elastic  fibres  or  connective-tissue  fibrils. 

Those  cells  that  offer  great  resistance  to  the  action  of  alkalies  and 
acids  have  not  been  carefully  studied.  According  to  former  views,  the 
matrix  was  considered  as  consisting  of  a  body  analogous  to  collagen,  so- 
called  chondrigen.  The  recent  investigations  of  Morochowetz  and  others, 
but  especially  those  of  C.  Th.  Morxer,1  have  shown  that  the  matrix  of 
the  cartilage  consists  of  a  mixture  of  collagen  with  other  bodies. 

The  tracheal,  thyroideal,  cricoidal,  and  arytenoidal  cartilages  of  full- 
grown  cattle  contain,  according  to  Morner,  four  constituents  in  the  matrix, 
namely,  chondromucoid,  chondroitin-sulphuric  acid,  collagen,  and  an  albumin- 
oid. 

Chondromucoid.  This  body,  according  to  Morxer,  has  the  composition 
C  47.30,  H  6.42,  N  12.58,  S  2.42,  O  31.28  per  cent.  Sulphur  is  in  part 
loosely  combined  and  may  be  split  off  by  the  action  of  alkalies,  and  a  part 
separates  as  sulphuric  acid  when  boiled  with  hydrochloric  acid.  Chondro- 
mucoid is  decomposed  by  dilute  alkalies  and  yields  alkali  albuminate,  pep- 
tone substances,  chondroitin-sulphuric  acid,  alkali  sulphides,  and  some 
alkali  sulphates.  On  boiling  with  acids  it  yields  acid  albuminate,  peptone 
substances,  chondroitin-sulphuric  acid,  and  on  account  of  the  further  de- 
composition of  this  last  body  sulphuric  acid  and  a  reducing  substance  are 
formed. 

Chondromucoid  is  a  white,  amorphous,  acid-reacting  powder  which  is 
insoluble  in  water,  but  dissolves  easily  on  the  addition  of  a  little  alkali. 
This  solution  is  precipitated  by  acetic  acid  in  great  excess  and  by  small 
quantities  of  mineral  acids.  The  precipitation  may  be  retarded  by  neutral 
salts  or  by  chondroitin-sulphuric  acid.  The  solution  containing  XaCl  and 
acidified  with  HC1  is  not  precipitated  by  potassium  ferrocyanide.  Precipi- 
tants  for  chondromucoid  are  alum,  ferric  chloride,  sugar  of  lead,  or  basic  lead 
acetate.  Chondromucoid  is  not  precipitated  by  tannic  acid,  and  it  may  by 
its  presence  prevent  the  precipitation  of  gelatine  by  this  acid.  It  gives  the 
usual  color  reactions  for  proteids,  namely,  with  nitric  acid,  with  copper 
sulphate  and  alkali,  with  Millon's  and  Adamkiewicz 's  reagents. 

Chondroitin-sulphuric  Acid,  chondroitic  acid.  This  acid,  which  was 
first  prepared  pure  from  cartilage  by  C.  Th.  Morxer  and  identified  by 

1  Morochowetz,  Verhandl.  d.  naturh.  med.  Vereins  zu  Heidelberg,  1,  Heft  5;  Morner, 
Skand.  Arch.  f.  physiol.,  1. 


CIIONDROITIN-S 1 1LPB  URIC   A  CID.  36 1 

him  as  an  ethereal  sulphuric  acid,  occurs,  according  to  Moknkr,  in  all  varie- 
ties of  cartilage  and  also  in  the  tunica  iutima  of  the  aorta  ami  as  traces 
in  the  bone  substance.  K.  Morner  has  also  found  it  in  the  ox-kidney 
ami  in  human  urine  as  a  regular  constituent.  According  to  Krawkow, 
who  found  it  in  the  cervical  ligament  of  the  ox,  it  combines  with  proteid, 
forming  amyloid  (see  page  54),  which  explains  the  occurrence  of  this  body 
in  amyloid  degenerated  livers,  as  observed  by  Oddi.1  The  identity  of  the 
ethereal  sulphuric  acid  occurring  in  liver  amyloid  with  chondroitin-sul- 
phuric  acid  does  not  seem  to  be  quite  clear,  according  to  the  researches  of 
Mi>\i;:ry.  According  to  Levene  3  the  glucothionic  acid,  prepared  from 
tendon  mucoid  and  which  gives  the  orcin  reaction  for  glucuronic  acid,  and 
yields  furfurol  on  distillation  with  hydrochloric  acid,  is  not  identical  with 
the  chondroitin-sulphuric  acid,  hence  more  acids  related  to  it  are  possible. 
Chondroitin-sulphuric  acid  has  the  formula  C18H27NS017,  according  to 
SniMiEDEBERG.3  As  first  products  this  acid  yields  on  cleavage  sulphuric 
acid  and  a  nitrogenous  substance,  chondroitin,  according  to  the  following 
equation : 

C18H27NS017  +H20  =  H2S04  +C18H27NOH. 

Chondroitin,  which  is  similar  to  gum  arabic  and  which  is  a  monobasic  acid, 
yields  acetic  acid  and  a  new  nitrogenous  substance,  chondrosin,  as  cleavage 
products,  on  decomposition  with  dilute  mineral  acids: 

C18H27N014  +  3H20  =  3C2H402  +  C12H21NOu. 

Chondrosin,  which  is  also  a  gummy  substance  soluble  in  water,  is  a  mono- 
basic acid  and  reduces  copper  oxide  in  alkaline  solution  even  more  strongly 
than  dextrose.  It  is  dextrogyrate  and  represents  the  reducing  substance 
obtained  by  previous  investigators  in  an  impure  form  on  boiling  cartilage 
with  an  acid.  The  products  obtained  on  decomposing  chondrosin  with 
barium  hydrate  tend  to  show,  according  to  Schmiedeberg,  that  chondro- 
sin contains  the  atomic  groups  of  glucuronic  acid  and  glucosamine.  This 
assumption  does  not  seem  to  have  sufficient  foundation.  According  to 
Ogler  and  Neuberg  4  chondrosin  does  not  give  the  orcin  test  nor  does 
it  yield  furfurol.  It  contains  neither  glucuronic  acid  nor  glucosamine,  and 
on  cleavage  with  baryta  it  yields,  besides  a  carbohydrate  complex  which 
has  not  been  studied,  an  oxyamino  acid  having  the  formula  C6H130BX;  also 
a  hexosamine  acid  or  tetraoxyaminocaproic  acid. 

1  C.  Morner,  1.  c,  and  Zeitschr.  f.  physiol.  Chem.,  20  and  23;    K.  Morner,  Skand. 
Arofe.  f.  Physiol.,  6;  Krawkow,  Arch.  f.  exp.  Path.  u.  Pharm.,  40;  oddi    ,/>,</.,  33. 
2Monorv,  Compt.  rend.  soc.  biol.,  54;   Levene,  Zeitschr.  f.  physiol.  Chem.,  39. 

3  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 

4  Zeitschr.  f.  physiol.  Chem.,  37. 


362  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

Chondroitin-sulphuric  acid  appears  as  a  white  amorphous  powder, 
which  dissolves  very  easily  in  water,  forming  an  acid  solution  and,  when 
sufficiently  concentrated,  a  sticky  liquid  similar  to  a  solution  of  gum  arabic. 
Nearly  all  of  its  salts  are  soluble  in  water.  The  neutralized  solution  is 
precipitated  by  tin  chloride,  basic  lead  acetate,  neutral  ferric  chloride, 
and  by  alcohol  in  the  presence  of  a  little  neutral  salt.  The  solution,  on 
the  other  hand,  is  not  precipitated  by  acetic  acid,  tannic  acid,  potassium 
ferrocyanide  and  acid,  sugar  of  lead,  mercuric  chloride,  or  silver  nitrate. 
Acidified  solutions  of  alkali  chondroitin-sulphates  cause  a  precipitation 
when  added  to  solutions  of  gelatine  or  proteid. 

Chondromucoid  and  chondroitin-sulphuric  acid  may  be  prepared,  accord- 
ing to  Morner,  by  extracting  finely  cut  cartilage  with  water,  which  dis- 
solves the  preformed  chondroitin-sulphuric  acid  besides  some  chondro- 
mucoid. In  this  watery  extract  the  chondroitin-sulphuric  acid  prevents 
the  precipitation  of  the  chondromucoid  by  means  of  an  acid.  If  2-4  p.  m. 
HC1  is  added  to  this  watery  extract  and  warmed  on  the  water-bath,  the 
chondromucoid  gradually  separates,  while  the  chondroitin-sulphuric  acid 
and  the  rest  of  the  chondromucoid  remain  in  the  filtrate.  If  the  cartilage, 
which  has  been  lixiviated,  at  the  temperature  of  the  body,  with  water,  is 
extracted  with  hydrochloric  acid  of  2-3  p.  m.  until  the  collagen  is  con- 
verted into  gelatine  and  dissolved,  the  remaining  chondromucoid  may  be 
removed  from  the  insoluble  residue  by  dilute  alkali  and  precipitated  from 
the  alkaline  extract  by  an  acid.  It  may  be  purified  by  repeated  solution 
in  water  with  the  aid  of  a  little  alkali,  by  precipitation  with  an  acid  and 
then  finally  treating  with  alcohol  and  ether. 

The  pre-existing  chondroitin-sulphuric  acid,  or  that  formed  by  the 
decomposition  of  chondromucoid,  is  obtained  by  lixiviating  the  cartilage 
with  a  5  per  cent  caustic-alkali  solution.  The  alkali  albuminate  formed 
by  the  decomposition  of  the  chondromucoid  can  be  removed  from  the 
solution  by  neutralization,  then  the  peptone  precipitated  by  tannic  acid, 
the  excess  of  this  acid  removed  with  sugar  of  lead,  and  the  lead  separated 
from  the  filtrate  by  H2S.  If  further  purification  is  necessary,  the  acid  is 
precipitated  with  alcohol,  the  precipitate  dissolved  in  water,  this  solution 
dialyzed  and  precipitated  again  with  alcohol, — this  solution  in  water  and 
precipitation  with  alcohol  being  repeated  a  few  times, — and  lastly  the  acid 
is  treated  with  alcohol  and  ether. 

Schmiedeberg  prepared  the  acid  from  the  septum  narium  of  the  pig 
according  to  the  following  method:  The  finely  divided  cartilage  is  first 
exposed  to  artificial  peptic  digestion,  then  carefully  washed  with  water 
and  the  insoluble  residue  treated  with  2-3  per  cent  hydrochloric  acid. 
This  cloudy  liquid  containing  hydrochloric  acid  is  precipitated  with  alcohol 
(about  \  vol.)  and  the  clear  filtrate  treated  with  absolute  alcohol  and 
some  ether.  The  precipitate,  consisting  chiefly  of  a  combination  or  a 
mixture  of  chondroitin-sulphuric  acid  and  gelatine  peptone  (pepto-chondrin), 
Is  first  washed  with  alcohol  and  then  with  water.  It  is  then  dissolved  in 
alkaline  water  and  the  basic  alkali  combination  precipitated  from  this 
solution  by  the  addition  of  alcohol,  whereby  the  gelatine-peptone  alkali 
remains  in  solution.  The  precipitate  is  purified  by  repeated  solution  in 
alkaline  water  and  precipitated  by  alcohol.     To  obtain  chondroitin-sul- 


COLLAGEN  AND  ALBUMINOID  OF  THE  CARTILAGE. 

phuric  acid  entirely  free  from  chondroitin  it  La  more  advantageous  to  pre- 
pare the  potassium-copper  combination  of  the  acid  from  the  alkaline  solu- 
tion by  the  alternate  addition  of  copper  acetate  and  caustic  potash  and 
precipitating  with  alcohol.  The  reader  is  referred  to  the  original  article 
for  more  details  and  also  for  Oddi's  method. 

The  collagen  of  the  cartilage  gives,  according  to  Morner,  a  gelatine  which 
contains  only  16. 4  per  cent  N  and  which  can  hardly  be  considered  identical 
with  ordinary  gelatine. 

In  the  above-mentioned  cartilages  of  full-grown  animals  the  chondroitin- 
sulphuric  acid  and  ehrondromucoid,  perhaps  also  the  collagen,  are  found 
surrounding  the  cells  as  round  balls  or  lumps.  These  balls  (Morner's 
chonrfrin-halls),  which  give  a  blue  color  with  methyl-violet,  lie  in  the  meshes 
of  a  trabecular  structure,  which  is  colored  when  brought  in  contact  with 
tropseolin. 

The  albuminoid  is  a  nitrogenized  body  which  contains  loosely  com- 
bined sulphur.  It  Is  soluble  with  difficulty  in  acids  and  alkalies  and 
resembles  keratin  in  many  respects,  but  differs  from  it  by  being  soluble 
in  gastric  juice.  In  other  respects  it  is  more  similar  to  elastin,  but  differs 
from  this  substance  by  containing  sulphur.  This  albuminoid  gives  the 
color  reactions  of  the  proteid  bodies. 

The  preparation  of  cartilage  gelatine  and  albuminoid  may  be  performed 
according  to  the  following  method  of  Morner:  First  remove  the  chon- 
dromucoid  and  chondroitin-sulphuric  acid  by  extraction  with  dilute  caustic- 
potash  (0.2-0.5  per  cent),  remove  the  alkali  from  the  remaining  cartilage 
by  water,  and  then  boil  with  water  in  a  Papin's  digester.  The  collagen 
passes  into  solution  as  gelatine,  while  the  albuminoid  remains  undissol\»ed 
(contaminated  by  the  cartilage-cells).  The  gelatine  may  be  purified  by 
precipitating  with  sodium  sulphate,  which  must  be  added  to  saturation  in 
the  faintly  acidified  solution,  redissolving  the  precipitate  in  water,  dialyzing 
well,  and  precipitating  with  alcohol. 

According  to  Morner,  no  albuminoid  is  found  in  young  cartilage,  but 
only  the  three  first-mentioned  constituents.  Nevertheless  the  young  carti- 
lage contains  about  the  same  amounts  of  nitrogen  and  mineral  substances 
as  the  old.  The  cartilage  of  the  ray  {Raja  batis  Lin.),  which  has  been 
investigated  by  Lonnberg,1  contains  no  albuminoid  and  only  a  little 
chondromucoid,  but  a  large  proportion  of  chondroitin-sulphuric  acid  and 
collagen. 

According  to  Pfluger  and  Handel  2  glycogen  occurs  to  a  slight  extent 
in  all  matrices,  and  of  these  it  is  richest  in  the  cartilage.  Tendons,  ligamen- 
tum  nuchea,  and  cartilage  of  the  ox  contained  0.06,  0.07,  and  2.17  p.  m. 
glycogen  respectively  (Handel). 

Hoppe-Seyler  found  in  fresh  human  rib-cartilage  676.7  p.  m.  water, 
301.3  p.m.  organic  and  22  p.m.  inorganic  substance,  and  in  the  cartilage 
1  Maly's  Jahresber.,  19,  325.  ■  Pfluger's  Arch.,  92;  Handel,  ibid. 


364  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

of  the  knee-joint  735.9  p.  m.  water,  248.7  p.  m.  organic  and  15.4  p.  m. 
inorganic  substance.  Pickardt  1  found  402-574  p.  m.  water  and  72.86  p.  m. 
ash  (no  iron)  in  the  laryngeal  cartilage  of  oxen.  The  ash  of  cartilage  con- 
tains considerable  amounts  (even  800  p.  m.)  of  alkali  sulphate,  which 
probably  does  not  exist  originally  as  such,  but  is  produced  in  great  part  by 
the  incineration  of  the  chondroitin-sulphuric  acid  and  the  chondromucoid. 
The  analyses  of  the  ash  of  cartilage  therefore  cannot  give  a  correct  idea  of 
the  quantity  of  mineral  bodies  existing  in  this  substance.  The  cartilage  is 
richest  in  sodium jrf  all  the  tissues  of  the  body,  and  according  to  Bunge  x  the 
amount  of  Na  and  CI  is  greatest  in  young  animals.  In  1000  parts  of  carti- 
lage dried  at  120°  C,  Bunge  found  91.26  parts  Na20  in  the  shark,  33.98  in 
the  ox  embryo,  32.45  in  a  fourteen-day-old  calf,  and  26.4  in  a  ten-week- 
old  calf. 

The  Cornea. /The  corneal  tissue,  which  is  considered  by  many  investi-\ 
/  gators  to  be  related  to  cartilage  in  a  chemical  sense,  contains  traces  of  » 
(  proteid  and  a  collagen  as  chief  constituent/which  C.  Th.  Morner  2  claims 
contains  16.95  per  cent  N.     According  to  him  it  also  contains  a  mucoid 
which  has  the  composition  C  50.16,  H  6.97,  N  12.79,  and  S  2.07  per  cent. 
On  boiling  with  dilute  mineral  acid  this  mucoid  yields  a  reducing  sub- 
stance.   The  globulins  found  by  other  investigators  in  the  cornea  are  not 
derived  from  the  matrix,  according  to  Morner,  but  from  the  layer  of 
epithelium.     According   to   Morner,   Descemet's   membrane   consists   of 
membranin  (page  53),  which  contains  14.77  per  cent  N  and  0.90  per  cent  S. 
In   the   cornea  of  oxen  His3  found  758.3  p.  m.  water,  203.8  p.  m. 
gelatine-forming  substance,  28.4   p.  m.  other  organic    substance,  besides 
8.1  p.  m.  soluble  and  1.1  p.  m.  insoluble  salts. 

III.   Bone. 

The  bony  structure  proper,  when  free  from  other  formations  occurring 
in  bones,  such  as  marrow,  nerves,  and  blood-vessels,  consists  of  cells  and  a 
matrix. 

The  cells  have  not  been  closely  studied  in  regard  to  their  chemical  con- 
stitution. On  boiling  with  water  they  yield  no  gelatine.  They  contain  no 
keratin,  which  is  not  usually  present  in  the  body  structure  (Herbert 
Smith  4). 

The  matrix  of  the  bony  structure  contains  two  chief  constituents, 
namely,  an  organic  substance,  and  the  so-called  bone-earths,  lime-salts, 
enclosed  in  or  combined  with  it.     If  bones  are  treated  with  dilute  hydro- 

1  Hoppe-Seyler,  cited  from  Kiihne's  Lehrbuch  d.  physiol.  Chem.,  387;  Pickardt, 
Centralbl.  f.  Physiol.,  6,  735;   Bunge,  Zeitschr.  f.  physiol.  Chem.,  28. 

2  Zeitschr.  f.  physiol.  Chem.,  18. 

8  Cited  from  Gamgee,  Physiol    Chem.,  1880,  451. 
*  Zeitschr.  f.  Biologie,  19. 


BONES.  3G5 

chloric  acid  at  the  ordinary  temperature,  the  lime-salts  are  dissolved  and 
the  organic  substance  remains  as  an  elastic  mass,  preserving  the  shape 

\  of  the  bone. 

The  organic  matrix  consists  chiefly  of  ossein,  which  is  generally  con- 
sidered as  identical  with  the  collagen  of  the  connective  tissue.     It  also 

Lpontains,  as  Haw  ic  and  GlES  '  have  shown,  mucoid  and  albuminoid, ./"After 
the  removal  of  the  lime-salts  by  hydrochloric  acid  of  2-5  p.  m.  these  experi- 
menters were  able  to  extract  the  mucoid  by  one-half  saturated  lime-water 
and  to  precipitate  it  with  2  p.  m.  hydrochloric  acid.  After  the  removal 
of  the  osseomucoid  and  collagen  (by  boiling  with  water)  they  obtained 
the  albuminoid  as  an  insoluble  residue. 

The  osseomucoid  on  boiling  with  hydrochloric  acid  yielded  a  reducing 
substance  and  sulphuric  acid,  1.11  per  cent  sulphur  appearing  in  this 
form.  The  osseomucoid  stands  close  to  the  chondro-  and  tendon  mucoid 
in  elementary  composition,  as  may  be  seen  from  the  following  analyses: 

c.  h.         n.  s.          o. 

Osseomucoid 47.43  6.63  12.22  2.32  31 .40  (Hawk  and  Gies) 

Chondromucoid 47.30  6.42  12.58  2.42  31.28  (C.  Morner) 

Tendon  mucoid 48.76  6.53  11.75  2.33  30.60  (Chittenden  and  Gies) 

Corneal  mucoid 50.16  6.97  12.79  2.07  28.01  (C.  Morner) 

The  osseoalbuminoid  is  insoluble  in  2  p.  m.  hydrochloric  acid  and  5  p.  m. 
Na^Og,  but  dissolves  in  10  per  cent  KOH  with  the  formation  of  albumin- 
ates.    The  composition  of  chondro-  and  osseoalbuminoid  is  as  follows: 

c.  h.  n.  s.  o. 

Osseoalbuminoid 50.16       7.03       16.17       1.18       25 .  46  )  Hawk  and 

Chondroalbuminoid 50.46       7.05       14.95       1.86       25.48)"         Gies 

The  inorganic  constituents  of  the  bony  structure,  the  so-called  bone-\ 
earths,  which  after  the  complete    calcination    of    the    organic    substance/ 
remain  as  a  white,  brittle  mass,  consist  chiefly  of  calcium  and  phosphoric 
acid,  but  also  contain  carbon  dioxide  and,  in  smaller  amounts,  magnesium,  i 
chlorine,  and  fluorine.     Alkali  sulphate  and  iron,  which  have  been  found 
in  bone-ash,  do  not  seem  to  belong  exactly  to  the  bony  substance,  but  to, 
the  nutritive  fluids  or  to  the  other  constituents  of  bones./  The  traces  of 
sulphate  occurring  in  the  bone-ash  are  derived,  according  to  Morner,2  from 
the   chondroitin-sulphuric    acid.     According    to   Gabriel  3  potassium  and 
sodium  are  essential  constituents  of  bone-earth. 

The  opinions  of  investigators  differ  somewhat  as  to  the  manner  in  which 
the  mineral  bodies  of  the  bony  structure  are  combined  with  each  other. 
Chlorine  is  present  in  the  same  form  as  in  apatite  (CaCl2,3Ca3P208).     If  we 


1  Amer.  Journ.  of  Physiol.,  5  and  7. 
'Zeitschr.  f.  physiol.  Chem.,  23. 
Ibid.,  18,  which  also  contains  the  pertinent  literature. 


365  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

eliminate  the  magnesium,  the  chlorine,  and  the  fluorine,  the  last,  according 
to  Gabriel,  occurring  only  as  traces,  the  remaining  mineral  bodies  form 
the  combination  3(Ca3P208)CaC03.  According  to  Gabriel  the  simplest  ex- 
pression for  the  composition  of  the  ash  of  bones  and  teeth  is  (Ca3(P04)2-f 
Ca5HP3013  +  Aq) ,  in  which  2-3  per  cent  of  the  lime  is  replaced  by  magnesia, 
potash,  and  soda,  and  4-6  per  cent  of  the  phosphoric  acid  by  carbon  dioxide, 
chlorine,  and  fluorine. 

'Analyses  of  bone-earths  have  shown  that  the  mineral  constituents  exist   I 
in  rather  constant  proportions,  which  is  nearly  the  same  in  different  animals.^ 
As  an  example  of  the  composition  of  bone-earth  we  here  give  the  analyses 
of  Zalesky.1    The  figures  represent  parts  per  thousand. 

Man.  Ox.  Tortoise.  Guinea-pig. 

Calcium  phosphate,  Ca3P208 838 . 9  860 . 9  859 . 8  873 . 8 

Magnesium  phosphate,  Mg3P2Os 10.4  10.2  13.6  10.5 

Calcium  combined  with  C02>  Fl,  and  CI...     76.5  73.6  63.2  70.3 

C02         57.3  62.0  52.7 

Chlorine. 1-8  2.0         1.3 

Fluorine2 2.3  3.0  2.0 

Some  of  the  C02  is  always  lost  on  calcining,  so  that  the  bone-ash  does  not 
contain  the  entire  C02  of  the  bony  substance. 

Ad.  Carnot  3  found  the  following  composition  for  the  bone-ash  of  man, 
ox,  and  elephant: 

Man.  Ox.  Elephant. 

Kd&  TS).  F—  Femur. 

Calcium  phosphate 874.5  878.7  857.2  900.3 

Magnesium  phosphate 15.7  17.5  15.3  19.6 

Calcium  fluoride 3.5  3.7  4.5  4.7 

Calcium  chloride 2.3  3.0  3.0  2.0 

Calcium  carbonate 101.8  92.3  119.6  72.7 

Iron  oxide 10  1.3  1.3  1.5 

The  quantity  of  organic  substance  in  the  bones,  calculated  from  the  loss 
of  weight  in  burning,  varies  somewhat  between  300  and  520  p.  m.  This 
variation  may  in  part  be  explained  by  the  difficulty  in  obtaining  the  bony 
substance  entirely  free  from  water  and  partly  by  the  very  variable  amount 
of  blood-vessels,  nerves,  marrow,  and  the  like  in  different  bones.  The 
unequal  amounts  of  organic  substance  found  in  the  compact  and  in  the 
spongy  parts  of  the  same  bone,  as  well  as  in  bones  at  different  periods 
of  development  in  the  same  animal,  depend  probably  upon  the  varying 
quantities  of  these  above-mentioned  tissues.  Dentin,  which  is  compara- 
tively pure  bony  structure,  contains  only  260-280  p.  m.  organic  substance, 
and  Hoppe-Seyler  4  therefore  thinks  it  probable  that  perfectly  pure  bony 


1  Hoppe-Sevler,  Med.-chem.  Untersuch.,  19. 

2  The  statements  as  to  the  quantity  of  fluorine  are  contradictory;;   see  Harms, 
Zeitschr.  f.  Biologie,  38;   Jodblauer,  ibid.,  41. 

3  Compt.  rend.,  114. 

« Physiol.  Chem.,  102-104. 


BONES.  367 

substance  lias  a  constant  composition  and  contains  only  about  250  p.  m. 
organic  substance.  The  question  whether  these  substances  are  chemically 
combined  with  the  bone-earths  or  only  intimately  mixed  has  not    been 

decided. 

The  nutritive  fluids  which  circulate  through  the  bones  have  not  been  isolated, 
and  we  only  know  that  they  contain  some  proteid  and  some  NaG  ami  alkali 
sulphate.  The  yellow  marrow  contains  chiefly  fat,  which  consists  of  olein,  pal- 
mitin,  and  stearin,  and  which  differs  from  the  fat  of  the  other  parts  of  the  body 
by  having  a  higher  acetyl  equivalent  (Zink  ').  Proteid  has  been  found  especially 
in  the  so-called  red  marrow  of  the  spongy  bones.  According  to  Forrest,  the 
proteid  consists  of  a  globulin  coagulating  at  47-50°  C.  and  a  Qucleo-albumin 
with  1.0  per  cent  phosphorus  (Halliburton  2),  besides  traces  of  albumin.  Besides 
this  the  marrow  contains  so-called  extractive  bodies,  such  as  lactic  acid,  hypo- 
xanthine,  and   cholesterin,  but  mostly  bodies  of  an  unknown  character. 

The  diverse  quantitative  composition  of  the  various  bones  of  the  skeleton 
depends  probably  on  the  varying  quantities  of  other  tissues,  such  as 
marrow,  blood-vessels,  etc.,  which  they  contain.  The  same  reason  explains, 
to  all  appearances,  the  larger  quantity  of  organic  substance  in  the  spongy 
parts  of  the  bones  as  compared  with  the  more  compact  parts.  Schrodt  3 
has  made  comparative  analyses  of  different  parts  of  the  skeleton  of  the  same 
animal  (dog)  and  has  found  an  essential  difference.  The  quantity  of  water 
in  the  fresh  bones  varies  between  138  and  443  p.  m.  The  bones  of  the 
extremities  and  the  skull  contain  138-222,  the  vertebra  168-443,  and  the 
ribs  324-356  p.  m.  water.  The  quantity  of  fat  varies  between  13  and  269 
p.  m.  The  largest  amount  of  fat,  256-269  p.  m.,  is  found  in  the  long  tubular 
bones,  while  only  13-175  p.  m.  fat  is  found  in  the  small  short  bones.  The 
quantity  of  organic  substance,  calculated  from  fresh  bones,  was  150-300 
p.  m.,  and  the  quantity  of  mineral  substances  290-563  p.  m.  Contrary  to 
the  general  supposition  the  greatest  amount  of  bone-earths  was  not  found  in 
the  femur,  but  in  the  first  three  cervical  vertebra?.  In  birds  the  tubular 
bones  are  richer  in  mineral  substances  than  in  the  fiat  bones  (During),  and 
the  greatest  quantity  of  mineral  bodies  has  been  found  in  the  humerus 
(Hiller,  During  *). 

We  do  not  possess  trustworthy  statements  in  regard  to  the  composition 
of  bones  at  different  ages.  The  analyses  by  E.  Voit  of  bones  of  do^s  ami 
by  Brubacher  of  bones  of  children  apparently  indicate  that  the  skeleton 
becomes  poorer  in  water  and  richer  in  ash  with  increase  in  age.  Graffen- 
BERGER  5  has  found  in  rabbits  64-7£  years  old  that  the  bones  contained  only 
140-170  p.  m.  water,  while  the  bones  of  the  full-grown  rabbit  2    1  years  old 

*See  Chem.  Centralbl.,  1897,  I,  296. 
2  Forrest,  Journ.  of  Physiol.,  17;   Halliburton,  ibid.,  18. 
:  Cited  from  Maly  's  Jahresber. ,  6. 

4  Hiller,  cited  from  Maly's  Jahresber.,  14;   During,  Zeitschr.  f.  physiol.  Chem.,  23. 
sVoit,  Zeitschr.  f.   Biologie,  16;    Brubacher,  ibid.,  27;    Graffenberger  in  Maly's 
Jahresber.,  21. 


36S  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

contained  200-240  p.  m.     The  bones  of  old  rabbits  contain  more  carbon 
dioxide  and  less  calcium  phosphate. 

The  composition  of  bones  of  animals  of  different  species  is  but  little  known. 
The  bones  of  birds  contain,  as  a  rule,  somewhat  more  water  than  those  of  mam- 
malia, and  the  bones  of  fishes  contain  the  largest  quantity  of  water.  The  bones 
of  fishes  and  amphibians  contain  a  greater  amount  of  organic  substance.  The 
bones  of  pachyderms  and  cetaceans  contain  a  large  proportion  of  calcium  carbo- 
nate; those  of  granivorous  birds  always  contain  silicic  acid.  The  bone-ash  of 
amphibians  and  fishes  contains  sodium  sulphate.  The  bones  of  fishes  seem  to 
contain  more  soluble  salts  than  the  bones  of  other  animals. 

A  great  many  experiments  have  been  made  to  determine  the  exchange  of 
material  in  the  bones — for  instance,  with  food  rich  in  lime  and  with  food 
deficient  in  lime — but  the  results  have  always  been  doubtful  or  contradic- 
tory. The  attempts,  also,  to  substitute  other  alkaline  earths  or  alumina  for 
the  lime  of  the  bones  have  given  contradictory  results.1  On  the  adminis- 
tration of  madder  the  bones  of  the  animal  are  found  to  be  colored  red  after 
a  few  days  or  weeks;  but  these  experiments  have  not  led  to  any  positive 
conclusion  in  regard  to  the  growth  or  metabolism  in  the  bones. 

Under  pathological  conditions,  as  in  rachitis  and  softening  of  the  bones, 
an  ossein  has  been  found  which  does  not  give  any  typical  gelatine  on  boiling 
with  water.  Otherwise  pathological  conditions  seem  to  affect  chiefly  the 
quantitative  composition  of  the  bones,  and  especially  the  relationship  be- 
tween the  organic  and  the  inorganic  substance.  In  exostosis  and  osteo- 
sclerosis the  quantity  of  organic  substance  is  generally  increased.  In  rachitis 
and  osteomalacia  the  quantity  of  bone-earths  is  considerably  decreased. 
Attempts  have  been  made  to  produce  rachitis  in  animals  by  the  use  of  food 
deficient  in  lime.  From  experiments  on  fully  developed  animals  contradic- 
tory results  have  been  obtained.  In  young,  undeveloped  animals  Erwin 
Voit  2  produced,  by  lack  of  lime-salts,  a  change  similar  to  rachitis.  In  full- 
grown  animals  the  bones  were  changed  after  a  long  time  because  of  the  lack 
of  lime-salts  in  the  food,  but  did  not  become  soft,  only  thinner  (osteoporosis). 
The  experiments  of  removing  the  lime-salts  from  the  bones  by  the  addition 
of  lactic  acid  to  the  food  have  led  to  no  positive  results  (Heitzmann,  Heiss, 
Baginsky  3).  Weiske,  on  the  contrary,  has  shown,  by  administering  dilute 
sulphuric  acid  or  monosodium  phosphate  with  the  food  (presupposing  that 
the  food  gave  no  alkaline  ash)  to  sheep  and  rabbits,  that  the  quantity  of 
mineral  bodies  in  the  bones  might  be  diminished.  On  feeding  continuously 
for  a  long  time  with  a  food  which  yielded  an  acid  ash  (cereal  grains)  Weiske 
has  observed  a  diminution  in  the  mineral  substances  of  the  bones  in  full- 


1  See  H.  Weiske,  Zeitschr.  f.  Biologie,  31. 

2  Zeitschr.  f.  Biologie,  16. 

'Heitzmann,  Maly's  Jahresber.,  3,  229;  Heiss,  Zeitschr.  f.  Biologie,  12;  Baginsky, 
Virchow's  Arch.,  87. 


BONES.  369 

grown  herbivora.1  A  few  investigators  are  of  the  opinion  that  in  rachitis,  as 
in  osteomalacosis,  a  solution  of  the  lime-salts  by  means  of  lactic  acid  takes 
place.  This  was  BUggested  by  the  fact  that  (J.  Weber  and  C.  Schmidt2 
found  lactic  acid  in  the  cyst-like,  altered  bony  substance  in  osteomalacia. 

Well-known  investigators  have  disputed  the  possibility  of  the  lime-salts 
being  washed  from  the  bones  in  osteomalacosis  by  means  of  lactic  acid.  They 
have  given  special  prominence  to  the  fact  that  the  lime-salts  held  in  solution 
by  the  lactic  acid  must  be  deposited  on  neutralization  of  the  acid  by  the 
alkaline  blood.  This  objection  is  not  very  important,  as  the  alkaline  blood- 
serum  has  the  property  to  a  high  degree  of  holding  earthy  phosphates  in 
solution,  which  fact  can  be  easily  proved.  The  investigations  of  Levy  3  con- 
tradict the  statement  as  to  the  solution  of  the  lime-salts  by  lactic  acid  in 
osteomalacia.  He  has  found  that  the  normal  relationship  6P04 :  lOCa  is 
retained  in  all  parts  of  the  bones  in  osteomalacia,  which  would  not  be  the 
case  if  the  bone-earths  were  dissolved  by  an  acid.  The  decrease  in  phos- 
phate occurs  in  the  same  quantitative  relationship  as  the  carbonate,  and 
according  to  Levy  in  osteomalacia  the  exhaustion  of  the  bone  takes  place 
by  a  decalcification  in  which  one  molecule  of  phosphate  carbonate  after  the 
other  is  removed. 

In  rachitis  the  quantity  of  organic  matter  has  been  found  to  vary  between  664 
and  811  p.  m.  The  quantity  of  inorganic  substance  was  189-336  p.  m.  These 
figures  refer  to  the  dried  substance.  According  to  Brubacher  rachitic  bones 
are  richer  in  water  than  the  bones  of  healthy  children,  and  poorer  in  mineral  bodies, 
especially  calcium  phosphate.  In  opposition  to  rachitis,  osteomalacosis  is  often 
characterized  by  the  conside-able  amount  of  fat  in  the  bones,  230-290  p.m.; 
but  as  a  rule  the  composition  varies  so  much  that  the  analyses  are  of  little  value. 
In  a  case  of  osteomalacosis  Chabkie  4  found  a  larger  quantity  of  magnesium 
than  calcium  in  a  bone.  The  ash  contained  417  p.  m.  phosphoric  acid,  222  p.  m. 
lime,  269  p.  m.  magnesia,  and  86  p.  m.  carbon  dioxide. 

The  tooth-structure  is  nearly  related,  from  a  chemical  standpoint,  to  I 
the  bony  structure. 

Of  the  three  chief  constituents  of  the  teeth — dentin,  enamel,  and 
cement — the  cement  is  to  be  considered  as  true  bony  structure,  and  as 
such  has  already  been  discussed  to  some  extent.  Dentin  has  the  same 
composition  as  the  bony  structure,  but  contains  somewhat  less  water.  The 
organic  substance  yields  gelatine  on  boiling;  but  the  dental  tubes  are 
not  dissolved,  therefore  they  cannot  consist  of  collagen.  In  dentin  260-280 
p.  m.  organic  substance  has  been  found.  Enamel  is  an  epithelium  forma- 
tion  containing   a  large  proportion   of  lime-salts.     Corresponding   to   its 

'Sec  Italy's  Jahresber.,  22;  also  Weiske,  Zeitschr.  f.  physiol.  Chem.,  20,  and 
Zeitschr.  f.  Biologie,  31. 

'Cited  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem.,  4.  Aufl. 

'Zeitschr.  f.  physiol.  Chem.,  19 

4Chabrie\  "Les  phenomenes  chim.  de  rossification,"  Paris,  1895,  65. 


S70  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

character  and  origin  the  organic  substance  of  the  enamel  does  not  yield  any 
gelatine.  Completely  developed  enamel  contains  the  least  water,  the  great- 
est quantity  of  mineral  substances,  and  is  the  hardest  of  all  the  tissues  of 
the  body.  In  full-grown  animals  it  contains  hardly  any  water,  and  the? 
quantity  of  organic  substance  amounts  to  only  20-40-68  p.imV  The  rela- 
tive amounts  of  calcium  and  phosphoric  acid  are,  according  to  the  analyses 
of  Hoppe-Seyler,  about  the  same  as  in  bone-earths.  The  quantity  of 
chlorine  according  to  Hoppe-Seyler  is  remarkably  high,  0.3-0.5  per 
cent,  while  Bertz  1  found  that  the  ash  of  enamel  was  free  from  chlorine 
and  that  dentin  was  very  poor  in  chlorine. 

Carnot,2  who  has  investigated  the  dentin  from  elephants,  has  found  4.3  p.  m. 
calcium  fluoride  in  the  ash.  In  ivory  he  found  only  2.0  p.  m.  Dentin  from 
elephants  is  rich  in  magnesium  phosphate,  which  is  still  mor  i  abundant  in  ivory. 

According  to  Gabriel  the  amount  of  fluorine  is  very  small  and  amounts 
to  1  p.  m.  in  ox-teeth.  It  is  no  greater  in  the  teeth  and  enamel  than  in 
the  bones.3  The  same  investigator  found  that  the  phosphates  are  strikingly 
small  in  the  enamel,  and  in  the  teeth  considerable  lime  is  replaced  by  mag- 
nesia. This  coincides  with  Bertz's  findings,  that  dentin  contains  twice  as 
much  magnesia  as  the  enamel. 

IV.  The  Fatty  Tissue. 

>^  The  membranes  of  the  fat-cells  withstand  the  action  of  alcohol  and 
ether.  They  are  not  dissolved  by  acetic  acid  nor  by  dilute  mineral  acids, 
but  are  dissolved  by  artificial  gastric  juice.  They  may  possibly  consist  of  a 
substance  closely  related  to  elastin.  The  fat-cells  contain,  besides  fat,  a 
yellow  pigment  which  in  emaciation  does  not  disappear  so  rapidly  as  the 
fat;  and  this  is  the  reason  that  the  subcutaneous  cellular  tissue  of  an 
emaciated  corpse  has  a  dark  orange-red  color.  The  cells  deficient  in  or 
nearly  free  from  fat,  which  remain  after  the  complete  disappearance  of  the 
latter,  seem  to  have  an  albuminous  protoplasm  rich  in  water. 

The  less  water  the  fatty  tissue  contains  the  richer  it  is  in  fat.  Schulze 
and  Reinecke  4  found  in  1000  parts 

Water.  Membrane.               Fat. 

Fatty  tissue  of  oxen 99 . 7  16 . 6  883 . 7 

"         "      "sheep 104.8  16.4  878.8 

"         "      "pigs 64.4  13.6  922.0 

//  The  fat  contained  in  the  fat^cells  consists  chiefly  of  triglycerides  of  M 
f  stearic,  palmitic,  and  oleic  acids.  Besides  these,  especially  in  the  less  solid  J 
\  kinds  of  fats,  there  are  glycerides  of  other  fatty  acids.     (See  Chapter  IV.) 

1  See  Maly's  Jahresber.,  30. 

2  Compt.  rend.,  114. 

8  See  foot-note  2.  page  366. 

4  Annal.  d.  Chem.  u.  Pharm.,  142. 


( 


ORICIS  OF   THE  FAT  OF   THE  BODY.  371 

In  all  animal  fats  there  are  besides  these,  as  Fn.   I  I<h-\i  \\\  '  has  shown, 
also  free,  non-volat ile  fatty  acids,  although  in  very  small  amounts. 

(  Human  fat  is  relatively  rich  in  olein,  the  quantity  in  the  subcutaneous 
fatty  tissue  being  70  s"  per  cent  or  more.9  In  new-born  infant-  it  is  poorer 
in  oleic  acid  than  in  adults  (KnOPFELMACHER,  SlEGERT,  JaECKLE);  the 
quantity  of  olein  increases  until  the  end  of  the  6rs1  year,  when  it  is  about 
the  same  as  in  adults^  The  composition  of  the  fat  in  man  as  well  as  in 
different  individuals  of  the  same  species  of  animals  is  rather  variable,  a  fact 
which  is  probably  dependent  upon  the  food.  According  to  the  researches  of 
Henriques  and  Bansen  the  fat  of  the  subcutaneous  fatty  tissue  is  richer 
in  olein  than  that  of  the  internal  organs;  this  has  also  been  observed  bv 
Leick.  and  WlNKLER.8  In  animals  with  a  thick  subcutaneous  fat  deposit 
the  outer  layers,  according  to  Henriques  and  Hansen,  are  richer  in  olein 
than  the  inner  layers.  The  fat  of  cold-blooded  animals  is  especially  rich 
in  olein.  The  fat  of  domestic  animals  has,  according  to  Amthor  and  Zink, 
a  less  oily  consistency  and  a  lower  iodine  and  acetyl  equivalent  than  the 
corresponding  fat  of  wild  animals.  Kreis  and  Hafxek  4  have  prepared 
a  mixture  of  glycerides  from  pig-fat,  which  contained  beside  two  stearic- 
acid  molecules  a  residue,  CnH^Oj,  which  is  daturic  acid  or  an  acid  isomeric 
with  it.  Under  pathological  conditions  the  fat  may  have  a  markedly 
pronounced  variation.  The  fat  of  lipoma  seems,  according  to  Jaeckle, 
to  be  poorer  in  lecithin  than  other  fats. 

The  properties  of  fats  in  general,  and  the  three  most  important  varieties 
of  fat,  have  already  been  considered  in  a  previous  chapter,  hence  the  forma- 
tion of  the  adipose  tissue  is  of  chief  interest  at  this  time. 

Tin  formation  of  fat  in  the  organism  may  occur  in  various  ways.  The 
fat  of  the  animal  body  may  consist  partly  of  absorbed  fat  of  the  food  de- 
posited in  the  tissues,  and  partly  of  fat  formed  in  the  organism  from  other 
bodies,  such  as  proteids  or  carbohydrates. 

That  the  fat  of  the  food  which  is  absorbed  in  the  intestinal  canal  may  be 
retained  by  the  tissues  has  been  shown  in  several  ways.  Radziejewski, 
Lebedefp,  and  Muxk  have  fed  dogs  with  various  fats,  such  as  linseed-oil, 
mutton-tallow,  and  rape-seed-oil,  and  have  afterwards  found  the  adminis- 
tered fat  in  the  tissues.  Hofmann  starved  dogs  until  they  appeared  to 
have  lost  their  fat  and  then  fed  them  upon  large  quantities  of  fat  and  only 
little  proteids.  When  the  animals  were  killed,  he  found  so  large  a  quantity 
of  fat  that  it  could  not  have  been  formed  from  the  administered  proteids   / 

1  Ludwig-Festechrift,  1874.     Leipzig. 

*  See  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  30  (literature). 

s  Knopfelmacher,  Jahrbuch  f.  Kinderheilkunde  (N.  F.),  45  (older  literature); 
Siegert,  Hofmeister's  Beitrage,  1;  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  30  (literature); 
Henriques  and  Hanson,  Skand.  Arch.  f.  Physiol.,  11;  Leick  and  Winkler,  Arch.  f. 
Path.  u.  rhann.,  4S. 

4Ber.  d.  d.  Chem.  Gesellsch..  3(5. 


372  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

alone,  but  the  greatest  part  must  have  been  derived  from  the  fat  of  the 
food.  Pettenkofer  and  VoiT  arrived  at  similar  results  in  regard  to  the 
behavior  of  the  absorbed  fats  in  the  organism,  though  their  experiments 
were  of  another  kind.  Munk  has  found  that  on  feeding  with  free  fatty 
acids  these  are  deposited  in  the  tissues,  not,  however,  as  such;  but  they 
are  transformed  by  synthesis  with  glycerine  into  neutral  fats  on  their  pas- 
sage from  the  intestine  into  the  thoracic  duct,  and  also  the  connection  between 
the  fat  of  the  food  and  of  the  body  has  been  shown  by  others,  especially 
Rosenfeld.  Coronedi  and  Marchetti  and  especially  Winternitz  ' 
have  recently  shown  that  the  iodized  fat  is  taken  up  in  the  intestinal  tract 
and  deposited  in  the  various  organs. 

f^Troteids  and  carbohydrates  are  considered  as  the  mother-substances  of 

I  the  fats  formed  in  the  organism. 

The  formation  of  the  so-called  corpse-wax,  adipocere,  which  consists  of  a. 
mixture  of  fatty  acids,  ammonia,  and  lime-soaps,  from  parts  of  the  corpse 
rich  in  proteids,  is  sometimes  given  as  a  proof  of  the  formation  of  fats  from, 
proteids.  The  accuracy  of  this  view  has,  however,  been  disputed,  and 
many  other  explanations  of  the  formation  of  this  substance  have  been 
offered.  According  to  the  experiments  of  Kratter  and  K.  B.  Lehmann 
it  seems  as  if  it  were  possible  by  experimental  means  to  convert  animal  tissue 
rich  in  proteids  (muscles)  into  adipocere  by  the  continuous  action  of  water. 
Irrespective  of  this,  Salkowski  has  shown  recently  that  in  the  formation 
of  adipocere  the  fat  itself  takes  part  in  that  the  olein  decomposes  with 
the  formation  of  solid  fatty  acids;  still  it  must  be  considered  that  lower 
organisms  undoubtedly  take  part  in  its  formation.  The  production  of 
adipocere  as  a  proof  of  the  formation  of  fat  from  proteids  is  disputed  by 
many  investigators  for  this  and  other  reasons. 

Fatty  degeneration  is  another  proof  of  the  formation  of  fat  from  pro- 
teids. From  the  investigations  of  Bauer  on  dogs  and  Leo  on  frogs  it  was 
assumed  that,  at  least  in  acute  poisoning  by  phosphorus,  a  fatty  degenera- 

-  tion,  with  the  formation  of  fat  from  proteids,  takes  j)lace/  Pfluger  has 

'  raised  such  strong  arguments  against  the  older  researches' as  well  as  the  more 
recent  one  of  Polimanti,  who  claims  to  have  shown  the  formation  of  fat 
from  proteids  in  phosphorus  poisoning,  that  we  cannot  consider  the  forma- 
tion of  fat  as  conclusively  proved.  Recent  investigations  of  Athana.siu, 
Taylor,  and  especially  of  Rosenfeld,2  have  shown  that  in  these  instances 
no  new  formation  of  fat  from  proteid  took  place,  but  rather  a  fat  migration 
(Rosenfeld)  . 

1  Coronedi  and  Marchetti,  cited  by  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  24. 
A  review  of  the  literature  on  fat  formation  may  be  found  in  Rosenfeld:  Fettbildung, 
in  Ergebnisse  der  Physiologie,  1,  Abt.  I. 

2  Bauer,  Zeitschr.  f.  Biologie,  7;  Leo,  Zeitschr.  f.  physiol.  Chem.,  9;  Polimanti „ 
Pfluger 's  Arch. ,  70 ;  Pfluger,  ibid. ,  51  (literature  on  the  formation  of  fat  from  proteid)  and 
71;  Athanasiu,  ibid.,  7-4;  Taylor,  Journ.  Exp.  Medicine,  4;  see  also  foot-note  5,  page  2^1. 


FORMATION  OF  FAT  IN   THE  BODY.  373 

Another  more  direct  proof  for  the  formation  of  fat  from  proteids  has 
been  given  by  Hofmaxx.  He  experimented  with  fly-maggots.  A  num- 
ber of  these  were  killed  and  the  quantity  of  fat  determined.  The  remained 
were  allowed  to  develop  in  blood  whoso  proportion  of  fat  had  been  previ- 
ously determined,  and  after  a  certain  time  they  were  killed  and  analyzed. 
He  found  in  them  from  seven  to  eleven  times  as  much  fat  as  was  contained  in 
the  maggots  first  analyzed  and  the  blood  taken  together.  PFLUGER  '  has 
made  the  objection  that  a  considerable  number  of  lower  fungi  develop  in 
the  Mood  under  these  conditions,  in  whose  cell-body  fats  and  carbohydrates 
are  formed  from  the  different  constituents  of  the  blood  and  their  decompo- 
sition products,  and  that  these  serve  as  food  for  the  maggots. 

As  a  more  direct  proof  of  fat  formation  from  proteids  the  investigations  I 
of  Pettexkofer  and  Yoit  are  often  quoted.  These  investigators  fed  dogs 
with  large  quantities  of  meat  containing  the  least  possible  proportion  of  } 
fat,  and  found  all  of  the  nitrogen  in  the  excreta,  but  only  a  part  of  the 
carbon.  As  an  explanation  of  these  conditions  it  has  been  assumed  that 
the  proteid  of  the  organism  splits  into  a  nitrogenized  and  a  non-nitrog- 
enized  part,  the  former  changing  into  the  nitrogenized  final  product, 
urea,  and  like  products,  and  the  other,  on  the  contrary,  being  retained 
in  the  organism  as  fat  (Pettexkofer  and  Yoit). 

Pfluger  has  arrived  at  the  following  conclusion  by  an  exhaustive 
criticism  of  Pettexkofer  and  Yoit's  experiments  and  a  careful  recal- 
culation of  their  balance-sheet:  that  these  very  meritorious  investigations, 
which  were  continued  for  a  series  of  years,  were  subject  to  such  great 
defects  that  they  are  not  conclusive  as  to  the  formation  of  fat  from  pro- 
teids. He  especially  emphasizes  the  fact  that  these  investigators  started 
from  a  wrong  assumption  as  to  the  elementary  composition  of  the  meat, 
and  that  the  quantity  of  nitrogen  assumed  by  them  was  too  low  and  the 
quantity  of  carbon  too  high.  The  relationship  of  nitrogen  to  carbon  in 
meat  poor  in  fat  was  assumed  by  Yoit  to  be  as  1:3.68,  while,  according  to 
Pfluger  it  is  1:3.22  for  fat-free  meat  after  deducting  the  glvcogen,  and 
according  to  Rubxer  1 : 3.2S  without  deducting  the  glycogen./  On  recalcu- 
lation of  the  figures  using  these  coefficients,  Pfluger  has  arrived  at 
the  conclusion  that  the  assumption  as  to  the  formation  of  fat  from  proteids 
finds  no  support  in  these  experiments ./ 

In  opposition  to  these  objections  E.  Yoit  and  M.  Cremer  have  made 
new  feeding  experiments  to  show  the  formation  of  fat  from  proteids,  but 
the  proof  of  these  recent  investigations  has  been  denied  by  Pfluger.  On 
feeding  a  dog  on  meat  poor  in  fat  (containing  a  known  quantity  of  ether 
extractives,  glycogen,  nitrogen,  water,  ami  ash),  Kumagawa  2  could  not 
prove  the  formation  of  fat  from  proteids.     According  to  him  the  animal 

1  See  Rosenfeld,  Fettbildung,  Ergebnisse  der  Physiologie,  1,  Abt.  I. 
■Ibid. 


374  TISSUES  OF   THE  CONNECTIVE  SUBSTANCE. 

body  under  normal  conditions  has  not  the  power  of  forming  fat  from  pro- 
teid. 

Several  French  investigators,  especially  Chauveau,  Gautier,  and  Kauf- 
mann,1 consider  the  formation  of  fat  from  proteids  as  positively  proved. 
Kaufmann  has  recently  substantiated  this  view  by  a  method  which  will 
be  spoken  of  in  detail  in  Chapter  XVIII,  in  which  he  studied  the  nitro- 
gen elimination  and  the  respiratory  gas  exchange  in  conjunction  with  the 
simultaneous  formation  of  heat. 

As  we  are  agreed  that  carbohydrates  and  glycogen,  as  well  as  sugar,  can   . 
be  formed  from  proteids,  the  fact  cannot  be  denied  that  possibly  an  indirect  j 
formation  of  fat  from  proteids,  with  a  carbohydrate  as  an  intermediate  I 
step,  can  take  place.     The  possibility  of  a  direct  fat  formation  from  pro- 
teids without  the  carbohydrate  as  intermediary  must  also  be  generally  ad-J 
mitted,  although  such  a  formation  has  not  been  conclusively  proved^  y 

According  to  Chauveau  and  Kaufmann,  in  the  direct  formation  of  fat 
from  proteids  the  fat  is  formed,  besides  urea,  carbon  dioxide,  and  water, 
as  an  intermediary  product  in  the  oxidation  of  the  proteids,  while  Gautier 
considers  the  formation  of  fat  from  proteids  as  a  cleavage  without  taking  up 
oxygen.  Drechsel  2  has  called  attention  to  the  fact  that  the  proteid 
molecule  probably  originally  contains  no  radical  with  more  than  six  or 
nine  carbon  atoms.  If  fat  is  formed  from  proteid  in  the  animal  body, 
then,  according  to  Drechsel,  such  formation  is  not  a  splitting  off  of  fat 
from  the  proteids,  but  rather  a  synthesis  from  primarily  formed  cleavage 
products  of  proteids  which  are  deficient  in  carbon. 

The  formation  of  fat  from  carbohydrates  in  the  animal  body  was  first 
suggested  by  Liebig  This  was  combated  for  some  time,  and  until  lately 
it  was  the  general  opinion  that  a  direct  formation  of  fat  from  carbohydrates 
had  not  been  proved,  but  also  that  it  was  improbable.  The  undoubtedly 
great  influence  of  the  carbohydrates  on  the  formation  of  fat  as  observed 
and  proven  by  Liebig  was  explained  by  the  statement  that  the  carbo- 
hydrates were  consumed  instead  of  the  absorbed  fat  or  that  derived  from 
the  proteids,  hence  they  have  a  sparing  action  on  the  fat.  By  means  of  a 
series  of  nutrition  experiments  with  foods  especially  rich  in  carbohydrates, 
La wes  and  Gilbert,  Soxhlet,  Tscherwinsky,  Meissl  and  Stromer  (on 
pigs),  B.  Schultze,  Chaniewski,  E.  VoiTand  C.  Lehmann  (on  geese),  I.  Munk 
and  Rubner  and  Lummert  3  (on  dogs)  apparently  prove  that  a  direct  forma- 

1  Kaufmann,  Arch,  de  Physiol.  (5),  8,  where  the  works  of  Chauveau  and  Gautier 
are  cited. 

2Ladenburg's  Handworterbuch  der  Chem.,  3,  543. 

3  La  wes  and  Gilbert,  Phil.  Transactions,  1859,  part  2;  Soxhlet,  see  Maly 's  Jahresber., 
11,  51;  Tscherwinsky,  Landwirthsch.,  Versuchsstaat,  29  (cited  from  Maly's  Jahresber., 
13);  Meissl  and  Stromer,  Wien.  Sitzungsber.,  88,  Abth.  3;  Schultze,  Maly's  Jahresber., 
11,  47;  Chaniewski,  Zeitschr.  f.  Biologic,  20;  Voit  and  Lehmann,  see  C.  v.  Voit,  Sitz- 
ungsber. d.  k.  bayer.  Akad.  d.  Wissensch.,  1885;  I.  Munk,  Virchow's  Arch.,  101;  Rubner, 
Zeitschr.  f.  Biologie,  22;  Lummert,  Pfliiger's  Arch.,  71. 


FORMATION  OF  FAT.  375 

tion  of  fat  from  carbohydrates  docs  actually  occur.  The  processes  by 
which  this  formation  takes  place  arc  still  unknown.  As  the  carbohydrates 
do  not  contain  as  complicated  carbon  chains  as  the  fata,  the  formation  of 
fat  from  carbohydrates  must  consist  of  a  synthesis,  in  which  the  group 
CI IOir  is  converted  into  CH2;   also  a  reduction  must  occur. 

r  Analogous  to  Nencki  's  view  as  to  the  butyric-acid  fermentation,  when 
from  the  sugar  lactic  acid  is  formed  and  from  this  C02H2  and  acetaldehyde 
(C,H40)  are  produced,  and  from  this  latter,  by  the  union  of  two  molecules, 
butyric  acid  is  formed,  so  Magnus-Levy  '  attempts  to  explain  the  forma- 
tion of  fat  in  the  animal  body  from  carbohydrates  by  synthesis  from 
aldehyde  and  reduction.  He  considers  that  the  process  proceeds  in  the 
following  way:  (a)  9C3H803  =  9C2H40+9II2+C02  and  (&)  9C2H40+7H2 
=  C18H3a02  (stearic  acid)  +TH?Qj 

After  feeding  with  very  large  quantities  of  carbohydrates  the  relation- 
ship between  the  inspired  oxygen  and  the  expired  carbon  dioxide,  i.e.,  the 

CO 
respiratory  quotient  —^ ,  was  found  greater  than  1  in  certain  cases  (Han- 

riot  and  Richet,  Bleibtreu,  Kaufmann,  Laulanie  2).  This  is  explained 
by  the  assumption  that  the  fat  is  formed  from  the  carbohydrate  by  a  cleavage 
setting  free  carbon  dioxide  and  water  without  taking  up  oxygen.  This 
increase  in  the  respiratory  quotient  also  depends  in  part  on  the  increased 
combustion  of  the  carbohydrate. 

When  food  contains  an  excess  of  fat  the  superfluous  amount  is  stored 
up  in  the  fatty  tissue,  and  on  partaking  of  food  deficient  in  fat  this  accu- 
mulation is  quickly  exhausted.  There  is  perhaps  not  one  of  the  various  tis- 
sues that  decreases  so  much  in  starvation  as  the  fatty  tissue.  The  organism, 
then,  possesses  in  this  tissue  a  depot  where  there  is  stored  during  proper 
alimentation  a  nutritive  substance  of  great  importance  in  the  development 
of  heat  and  vital  force,  which  substance,  on  insufficient  nutrition,  is  given 
off  as  may  be  needed.  On  account  of  their  low  conducting  power  the  fatty- 
tissues  become  of  great  importance  in  regulating  the  loss  of  heat  from  the 
body.  They  also  serve  to  fill  cavities  and  as  a  protection  and  support  to 
\  certain  internal  organs. 

1Arch.  f.  (Anat.  u.)  Physiol.,  1901. 

1  Han  riot  and  Richet,  Annal.  de  Chim.  et  de  Pbys.  (6),  22;  Bleibtreu,  Pfliiger's 
Arch.,  56  and  So;  Kaufmann,  Arch,  de  Physiol.  (5),  8;  Laulanid,  ibid.,  791. 


\ 


CHAPTER  XI. 
MUSCLES. 

Striated  Muscles. 

In  the  study  of  the  muscles  the  chief  problem  for  physiological  chem- 
istry is  to  isolate  their  different  morphological  elements  and  to  investigate 
each  element  separately.  By  reason  of  the  complicated  structure  of  the 
muscles  this  has  been  thus  far  almost  impossible,  and  we  must  be  satisfied 
at  the  present  time  with  a  few  microchemical  reactions  in  the  investi- 
gation of  the  chemical  composition  of  the  muscular  fibres. 

Each  muscle-tube  or  muscle-fibre  consists  of  a  sheath,  the  sarcolemma, 
which  seems  to  be  composed  of  a  substance  similar  to  elastin,  and  contain- 
ing a  large  proportion  of  proteid.  This  last,  which  in  life  possesses  the 
power  of  contractility,  has  in  the  inactive  muscle  an  alkaline  reaction,  or, 
more  correctly  speaking,  an  amphoteric  reaction  with  a  predominating 
action  on  red  litmus  paper.  Rohmann  has  found  that  the  fresh,  inactive 
muscle  shows  an  alkaline  reaction  with  red  lacmoid,  and  an  acid  reaction 
with  brown  turmeric.  From  the  behavior  of  these  coloring-matters  with 
various  acids  and  salts  he  concludes  that  the  alkalinity  of  the  fresh  muscle 
with  lacmoid  is  due  to  sodium  bicarbonate,  diphosphate,  and  probably  also 
to  an  alkaline  combination  of  proteid  bodies,  and  the  acid  reaction  with 
turmeric,  on  the  contrary,  to  monophosphate  chiefly.  The  dead  muscle 
has  an  acid  reaction,  or  more  correctly  the  acidity  with  turmeric  increases 
on  the  decease  of  the  muscle,  and  the  alkalinity  with  lacmoid  decreases. 
The  difference  depends  on  the  presence  of  a  larger  quantity  of  monophos- 
phate in  the  dead  muscle,  and  according  to  Rohmann  free  lactic  acid  is 
found  in  neither  the  one  case  nor  the  other.1 

If  the  somewhat  disputed  statements  relative  to  the  finer  structure  of  the 
muscles  are  disregarded,  one  can  differentiate  in  the  striated  muscles  be- 
tween the  two  chief  components,  the  doubly  refracting — anisotropous — and 
the  singly  refracting — isotropous — substance.  If  the  muscular  fibres  are 
treated  with  reagents  which  dissolve  proteids,  such  as  dilute  hydrochloric 
acid,  soda  solution,  or  gastric  juice,  they  swell  greatly  and  break  up  into 

1  The  various  theories  in  regard  to  the  reaction  of  the  muscles  and  the  cause  thereof 
are  conflicting.  See  PaJhmann,  Pfluger's  Arch.,  50  and  55;  Heffter,  Arch.  f.  exp. 
Path.  u.  Pharm.,  31  and  38.     These  references  contain  the  pertinent  literature. 

376 


PR0TE1DS  OF   THE  MUSCLES.  377 

"Bowman's  disks."  By  the  action  of  alcohol,  chromic  acid,  boiling  water, 
or  in  general  such  reagents  as  cause  a  shrinking,  the  fibres  split  longitu- 
dinally into  fibrils;  and  this  behavior  shows  that  several  chemically  differ- 
ent substances  df  various  solubilities  enter  into  the  construction  of  the  mus- 
cular fibres. 

The  proteid  myosin  is  generally  considered  as  the  chief  constituent  of 
the  diagonal  disks,  while  the  isotropous  substance  contains  the  chief  mass 
of  the  other  proteids  of  the  muscles  as  well  as  the  chief  portion  of  the  ex- 
tractives. According  to  the  observations  of  Danilkwsky,  confirmed  by 
J.  Holmgren,1  myosin  may  be  completely  extracted  from  the  muscle  with- 
out changing  its  structure,  by  means  of  a  5  per  cent  solution  of  ammonium 
chloride,  which  fact  contradicts  the  above  view.  Danilewsky  claims  that 
another  proteid-like  substance,  insoluble  in  ammonium  chloride  and  only 
swelling  up  therein,  enters  essentially  into  the  structure  of  the  muscles. 
The  proteids,  which  form  the  chief  part  of  the  solids  of  the  muscles,  are  of 
the  greatest  importance. 

Proteids  of  the  Muscles. 

Like  the  blood  which  contains  a  fluid,  the  blood-plasma,  which  sponta- 
neously coagulates,  separating  fibrin  and  yielding  blood-serum,  so  also  the 
living  muscle,  at  least  of  cold-blooded  animals,  contains,  as  first  shown  by 
Kuhne,  a  spontaneously  coagulating  liquid,  the  muscle-plasma,  which  coagu- 
lates quickly,  separating  a  proteid  body,  myosin,  and  yielding  also  a  serum. 
That  liquid  which  is  obtained  by  pressing  the  living  muscle  is  called  muscle- 
plasma,  while  that  obtained  from  the  dead  muscle  is  called  muscle-serum. 
These  two  fluids  contain  different  proteid  bodies. 

Muscle-plasma  was  first  prepared  by  Kuhne  from  frog-muscles,  and 
later  by  Halliburton,  according  to  the  same  method,  from  the  muscles 
of  warm-blooded  animals,  especially  rabbits.  The  principle  of  this  method 
is  as  follows:  The  blood  Is  removed  from  the  muscles  immediately  after 
the  death  of  the  animal  by  passing  through  them  a  strongly  cooled  common- 
salt  solution  of  5-6  p.  m.  Then  the  muscles  are  quickly  cut  and  immediately 
thoroughly  frozen  so  that  they  can  be  ground  in  this  state  to  a  fine  mass — 
"muscle-snow."  This  pulp  is  strongly  pressed  in  the  cold,  and  the  liquid 
which  exudes  is  called  muscle-plasma.  According  to  v.  Furth  2  this  cooling 
or  freezing  is  not  necessary.  It  is  sufficient  to  extract  the  muscle  free  from 
blood,  as  above  directed,  with  a  6  p.  m.  common-salt  solution. 

1  Danilewsky,  Zeitschr.  f.  physiol.  Chem.,  ";  J.  Holmgren,  Maly'a  Jahresber.,  23. 

'See  Kuhne,  Untersuchungen  iiber  das  Protoplasma  (Leipzig.  1864),  2;  Hallibur- 
ton, Journ.  of  Phvsiol.,  S;  v.  Furth,  Arch.  f.  exp.  Path.  u.  Pharm.,  30  and  3";  Hof- 
meister's  Reitriige,  3,  and  Ergebnisse  der  Physiologic,  1,  Abt.  I;  Stewart  and  Soll- 
mann.  Journ.  of  Physiol.,  24. 


378  MUSCLE. 

Muscle-plasma  forms  a  yellow  to  brownish-colored  fluid  with  an  akaline 
reaction.  It  is  somewhat  different  in  different  animals.  Muscle-plasma 
from  the  frog  spontaneously  coagulates  slowly  at  a  little  above  0°  C,  but 
quicker  at  the  temperature  of  the  body.  Muscle-plasma  from  mammals 
coagulates,  according  to  v.  Furth,  even  slowly  at  the  temperature  of  the 
room,  though  only  slightly,  and  it  can  hardly  be  considered  as  a  process 
comparable  with  the  coagulation  of  the  blood.  Indeed  the  question  may  be 
asked  whether  a  true  muscle-plasma  does  exist  in  warm-blooded  animals, 
or  whether  the  fluid  obtained  from  such  muscles  exactly  represents  the 
plasma  of  the  living  muscle.  According  to  Kuhne  and  v.  Furth  the  reac- 
tion remains  alkaline  during  coagulation,  while  according  to  Halliburton, 
Stewart  and  Sollmann,  it  becomes  acid.  According  to  the  older  views 
the  clot  consists  of  globulin  and  myosin,  while  v.  Furth  claims  that  it  con- 
sists of  two  coagulable  proteids,  myosin  fibrin  and  myogen  fibrin. 

The  study  of  the  proteids  of  the  muscles,  as  well  as  their  nomenclature, 
has  changed  markedly  in  the  last  few  years  and  it  is  questionable  whether 
an  essential  difference  exists  between  the  proteids  of  the  muscle-plasma  and 
the  muscle-serum  of  warm-blooded  animals.  Nevertheless  it  is  necessary 
to  separately  discuss  the  proteids  of  the  dead  muscles  as  well  as  those  o ' 
the  muscle-plasma. 

The  proteids  of  the  dead  muscle  are  in  part  soluble  in  water  or  dilute 
salt  solutions,  and  part  are  insoluble  therein.  Myosin  and  musculin  and 
also  myoglobulin  and  myoalbumin,  which  exist  to  a  very  slight  extent  and 
are  perhaps  only  derived  from  the  remaining  lymph,  belong  to  the  first 
group,  and  the  stroma  substances  of  the  muscle-tubes  belong  to  the  second 
group. 

Myosin  was  first  discovered  by  Kuhne,  and  constitutes  the  principal 
mass  of  the  soluble  proteids  of  the  dead  muscle,  and  is  generally  considered 
as  the  most  essential  coagulation  product  of  muscle-plasma.  With  the 
name  myosin  Kuhne  also  designates  the  mother-substance  of  the  plasma- 
clot,  and  this  mother-substance  forms,  according  to  certain  investigators, 
the  chief  mass  of  contractile  protoplasm.  The  statements  as  to  the  occur- 
rence of  myosin  in  other  organs  besides  the  muscles  require  further  proof. 
The  quantity  of  myosin  in  the  muscles  of  different  animals  varies,  accord- 
ing to  Danilewsky,1  between  30  and  110  p.  m. 

Myosin,  as  obtained  from  dead  muscles,  is  a  globulin  whose  elementary 
composition,  according  to  Chittenden  and  Cummins,2  is,  on  an  average, 
the  following:  C  52.82,  H  7.11,  N  16.17,  S.  1.27,  0  22.03  per  cent.  If  the 
myosin  separates  as  fibres,  or  if  a  myosin  solution  with  a  minimum  quantity 
of  alkali  is  allowed  to  evaporate  on  a  microscope-slide  to  a  gelatinous  mass, 
doubly  refracting  myosin  may  be  obtained.     Myosin  has  the  general  prop- 

1  Zeitschr.  f.  physiol.  Chem.,  7. 

2  Studies  from  the  Physiol.  Chcm.  Laboratory  of  Yale  College,  New  Haven,  3,  115. 


MYOSIN  AND  MUSCULW.  379 

erties  of  the  globulins.     It  is  insoluble  in  water,  but  soluble  in  dilute  Baline 

solutions  as  well  as  dilute  acids  or  alkalies,  which  readily  convert    it   into 

albuminates.  It  is  completely  precipitated  upon  saturation  with  NaCl,  also 
by  MgS< »,.  in  a  solution  containing  94  per  cent  of  the  salt  with  its  water  of 

crystallization  (  HALLIBURTON).     The  precipitated  myosin  becomes  insoluble 

readily,  bike  fibrinogen  it  coagulates  at  56°  C.  in  a  solution  containing 
common  salt,  but  differs  from  it  since  under  no  circumstances  can  it  be 
converted  into  fibrin.  The  coagulation  temperature,  according  to  Chitten- 
den and  CUMMINS,  not  only  varies  for  myosins  of  different  origin,  but  also 
for  the  same  myosin  in  different  salt  solutions. 

Myosin  may  be  prepared  in  the  following  way,  as  suggested  by  Halli- 
burton: The  muscle  is  first  extracted  by  a  5  per  cent  magnesium-sulphate 
solution.  The  filtered  extract  is  then  treated  with  magnesium  sulphate  in 
substance  until  100  c.  c.  of  the  liquid  contains  about  50  grams  of  the  salt. 
The  so-called  paramyosinogen  or  musculin  separates.  The  filtered  liquid 
is  then  treated  with  magnesium  sulphate  until  each  100  c.  c.  of  the  liquid 
holds  94  grams  of  the  salt  in  solution.  The  myosin  which  now  separates 
is  filtered  off,  dissolved  in  water  by  aid  of  the  retained  salt,  precipitated 
by  diluting  with  water,  and,  when  necessary,  purified  by  redissolving  in 
dilute  salt  solution  and  precipitating  with  water. 

The  older  and  perhaps  the  usual  method  of  preparation  consists,  accord- 
ing to  DANILEWSKY,1  in  extracting  the  muscle  with  a  5-10  per  cent  ammo- 
nium-chloride solution,  precipitating  the  myosin  from  the  filtrate  by 
strongly  diluting  with  water,  redissolving  the  precipitate  in  ammonium- 
chloride  solution,  and  the  myosin  obtained  from  this  solution  is  either 
reprecipitated  by  diluting  with  water  or  by  removing  the  salt  by  dialysis. 

Musculin,2  called  Paramyosinogen  by  Halliburton,  and  Myosin  by  v. 
Ff  rth,  is  a  globulin  which  is  characterized  by  its  low  coagulation  tempera- 
ture, about  47°  C,  which  may  vary  in  different  species  of  animal-  (  lo- 
in fro.LS,  51°. C.  in  birds).  It  is  more  easily  precipitated  than  myosin  by 
XaCl  or  MgS04  (salt  containing  50  per  cent  water  of  crystallization).  Ac- 
cording to  v.  Furth  it  is  precipitated  by  ammonium  sulphate  with  a  con- 
centration of  12-24  per  cent  salt.  If  the  dead  muscle  is  extracted  with 
water  a  part  of  the  musculin  goes  into  solution  and  may  be  precipitated 
therefrom  by  carefully  acidifying.  It  separates  from  a  dilute  salt  solution 
on  dialysis.  Musculin  readily  passes  into  an  insoluble  modification  which 
v.  FI'rtii  calls  myosin  fibrin.  Musculin  is  called  myosin  by  v.  Furtii.  as 
he  considers  it  nothing  but  myosin.  As  musculin  has  a  lower  coagulation 
temperature  and  has  other  precipitating  properties  for  neutral  salts  than 
the  older  substance  called  myosin,  it  is  difficult  to  concede  to  this  view. 

1  Zoitschr.  f.  phvsiol.  Chem.,  5,  15S. 

2  As  we  have  up  to  the  present  no  conclusive  basis  for  the  identity  of  the  globulins 
called  myosin  and  paramyosinogen,  and  also  as  the  use  of  the  name  myosin  for  the  List- 
mentioned  substance  may  readily  cause  confusion,  the  author  does  not  feel  justified 
in  dropping  the  old  name  musculin  (Xasse). 


3S0  MUSCLE. 

Myoglobulin.  After  the  separation  of  the  musculin  and  the  myosin  from  the 
salt  extract  of  the  muscle  by  means  of  MgS04  the  myoglobulin  may  be  precipitated 
by  saturating  the  filtrate  with  the  salt.  It  is  similar  to  serglobulin,  but  coagu- 
lates at  63°  C.  (Halliburton).  Myoalbumin,  or  muscle-albumin,  seems  to  be 
identical  with  seralbumin  (seralbumin  a,  according  to  Halliburton),  and  prob- 
ably only  originates  from  the  blood  or  the  lymph.  Proteoses  and  peptones  do 
not  seem  to  exist  in  the  fresh  muscles. 

After  the  complete  removal  from  the  muscle  of  all  proteid  bodies  which 
are  soluble  in  water  and  ammonium  chloride,  an  insoluble  proteid  remains 
which  only  swells  in  ammonium-chloride  solution  and  which  forms  with 
the  other  insoluble  constituents  of  the  muscular  fibre  the  "muscle-stroma." 
According  to  Danilewsky  the  amount  of  such  stroma  substance  is  con- 
nected with  the  muscle  activity.  He  maintains  that  the  muscles  contain 
a  greater  amount  of  this  substance,  compared  with  the  myosin  present, 
when  the  muscles  are  quickly  contracted  and  relaxed. 

According  to  J.  Holmgren  *  this  stroma  substance  does  not  belong  to 
either  the  nucleoalbumin  or  the  nucleoproteid  group.  It  is  not  a  gluco- 
proteid,  as  it  does  not  yield  a  reducing  substance  when  boiled  with  dilute 
mineral  acids.  It  is  very  similar  to  the  coagulable  proteids  and  dissolves 
in  dilute  alkalies,  forming  an  albuminate.  The  elementary  composition  of 
this  substance  is  nearly  the  same  as  that  of  myosin.  There  is  no  doubt 
that  the  insoluble  substances,  myofibrin  and  myosin  fibrin,  which  are 
formed,  according  to  v.  Furth,  in  the  coagulation  of  the  plasma,  occur  also 
among  the  stroma  substances.  When  the  muscles  are  previously  extracted 
writh  water  the  stroma  substance  also  contains  a  part  of  the  myosin  hereby 
made  insoluble.  To  the  proteids  insoluble  in  water  and  neutral  salts  belongs 
the  nucleoproteid  detected  by  Pekelharing,2  and  occurring  as  traces  and 
soluble  in  faintly  alkaline  water,  and  which  originates  probably  from  the 
muscle  nuclei.  According  to  Bottazzi  and  Ducceschi  the  heart  muscle  is 
richer  in  nucleoproteid  than  the  skeletal  muscle. 

Muscle-syntonin,  which  may  be  obtained  by  extracting  the  muscles  with  hydro- 
chloric acid  of  1  p.  m.,  and  which,  according  to  K.  Morner,  is  less  soluble  and 
has  a  greater  aptitude  to  precipitate  than  other  acid  albumins,  seems  not  to  occur 
preformed  in  the  muscles. 

Proteids  of  the  Muscle-plasma.  As  above  stated,  myosin  was  ordinarily 
considered  as  the  coagulated  modification  of  a  soluble  proteid  existing  in  the 
muscle-plasma.  As  in  blood-plasma  there  is  present  a  mother-substance  of 
fibrin,  fibrinogen,  so  also  there  exists  in  the  muscle-plasma  a  mother-substance 
of  myosin,  a  soluble  myosin  or  a  myosinogen.  This  body  has  not  thus  far 
been  isolated  with  certainty.   Halliburton,  who  has  detected  in  the  muscles 

1  See  foot-note  1,  page  377. 

'Pekelharing,  Zeitschr.  f.  physiol.  Chem.,  22;  Bottazzi  and  Ducceschi,  Centralbl. 
f.  Physiol.,  12. 


PRO TE IDS  OF   THE  MUSCLE  PLASMA.  381 

an  enzyme-like  substance,  "myosin  ferment,"  has  also  found  that  a  solu- 
tion of  purified  myosin,  in  dilute  salt  solution  (5  per  cent  MgS04),  and  suffi- 
ciently diluted  with  water,  coagulates  after  a  certain  time,  and  at  the  same 
time  becomes  acid,  and  a  typical  myosin-clot  separates.  This  coagulation, 
which  is  accelerated  by  warming  or  by  the  addition  of  myosin  ferment,  is, 
according  to  Halliburton,  a  process  analogous  to  the  coagulation  of  the 
muscle-plasma.  According  to  this  same  investigator,  myosin  when  dis- 
solved in  water  by  the  aid  of  a  neutral  salt  is  reconverted  into  myosinogen, 
while  after  diluting  with  water  myosin  is  again  produced  from  the  myosin- 
ogen. The  musculin  (paramyosinogen)  is  carried  down,  according  to  Hal- 
liburton', with  the  myosin-clot,  but  has  nothing  to  do  with  the  coagulation, 
as  the  myosin-clot  forms  also  in  the  absence  of  musculin,  and  this  last  is  not 
changed  into  myosin. 

Besides  the  traces  of  globulin  and  albumin,  which  perhaps  do  not  belong 
to  the  muscle-plasma,  there  occur  in  mammals,  according  to  v.  Furth:,  two 
proteids,  namely,  musculin  (myosin  according  to  v.  Furth)  and  myogen. 

Musculin  (Nasse)  —paramyosinogen  (Halliburton-)  =  myosin  (v. 
Furth)  forms  about  20  per  cent  of  the  total  proteids  of  the  muscle-plasma 
of  rabbits.  Its  properties  have  already  been  given,  and  it  is  sufficient  to 
remark  that  its  solutions  become  cloudy  on  standing,  and  a  precipitate  of 
myosin  fibrin  occurs,  which  is  insoluble  in  salt  solutions. 

Myogen,  or  myosinogen  (Halliburton),  forms  the  chief  mass,  75-80 
per  cent  of  the  proteids  of  rabbit-muscle  plasma.  It  does  not  separate  from 
lutions  on  dialysis  and  is  not  a  true  globulin,  but  a  proteid  sui  generis. 
It  coagulates  at  55-65°  C.  and  is  precipitated  in  the  presence  of  24-40  per 
cent  ammonium  sulphate.  Myogen  solutions  are  precipitated  by  acetic 
acid  only  in  the  presence  of  some  salt.  It  is  converted  into  an  albuminate 
by  alkalies,  this  albuminate  being  precipitable  by  ammonium  chloride. 
Myogen  passes  spontaneously,  especially  with  higher  temperatures  as  well 
as  in  the  presence  of  salt,  into  an  insoluble  modification,  myogen  fibrin.  A 
proteid,  coagulating  at  30— 10°  C  soluble  myogen  fibrin  is  produced  as  a  solu- 
ble intermediate  step.  This  substance  occurs  to  a  considerable  extent  in 
native  frog-muscle  plasma.  It  does  not  always  occur  in  the  muscle-plasma 
of  warm-blooded  animals,  and  when  it  does  it  is  present  only  to  a  slight 
extent.  It  can  be  separated  by  precipitating  with  salt  or  by  diffusion.  Hal- 
li  burton  's  assumption  as  to  the  action  of  a  special  myosin  ferment  has  not 
sufficient  basis,  according  to  v.  Furth,  nor  has  the  often-admitted  analogy 
With  the  coagulation  of  the  blood.  The  difference  between  the  musculin 
and  the  myogen  becoming  insoluble  is  that  the  musculin  passes  into  myosin 
fibrin  without  any  soluble  intermediate  steps. 

Myogen  may  be  prepared,  according  to  v.  Furth,  by  transiently  heating 
the  dialvzed  and  filtered  plasma  to  52°  C,  separating  it  in  this  way  from  the 
rest  of  the  musculin.     The  myogen  exists  in  the  new  filtrate  and  can  be 


3S2  MUSCLE. 

precipitated  by  ammonium  sulphate.  The  musculin  may  also  be  removed 
by  adding  28  per  cent  ammonium  sulphate  and  then  precipitating  the  myogen 
from  the  filtrate  by  saturating  with  the  salt. 

Stewart  and  Sollmann  admit  of  only  two  soluble  proteids  in  the  mus- 
cles. One  is  the  paramyosinogen,  which  is  the  same  as  v.  Furth  's  myosin  + 
the  soluble  myogenfibrin.  The  other  they  call  myosinogen,  which  corresponds 
to  v.  Furth 's  myogen  or  to  Halliburton's  myosinogen +myoglobulin. 
It  is  an  atypical  globulin  which  coagulates  at  50-60°  C.  The  paramyosinogen 
as  well  as  the  myosinogen  are  readily  converted  into  an  insoluble  modifi- 
cation, myosin.  The  myosin  of  the  above  investigators  is  the  same  as 
v.  Furth  's  myosin  fibrin  +  myogen  fibrin,  and  corresponds,  it  seems,  also  to 
myosin  mixed  with  paramyosinogen  (Halliburton).  Stewart  and  Soll- 
mann differ  from  Halliburton  in  considering  that  paramyosinogen  also 
coagulates  and  is  converted  into  myosin.  According  to  them  myosin  is 
also  insoluble  in  a  NaCl  solution. 

The  views  of  the  various  investigators  differ  so  essentially  and  the  nomen- 
clature is  so  complicated  (four  different  things  are  designated  by  the  name 
myosin)  that  it  is  extremely  difficult  to  give  any  correct  review  of  the  vari- 
ous notions.1    Thorough  investigations  on  this  subject  are  very  necessary. 

Myoproteid  is  a  proteid  found  by  v.  Furth  in  the  plasma  from  fish- 
muscles.  It  does  not  coagulate  on  boiling,  is  precipitated  by  acetic  acid, 
and  considered  as  a  compound  proteid  by  v.  Furth. 

In  connection  with  v.  Furth 's  work,  Przibram  has  carried  on  investigations 
on  the  occurrence  of  muscle  proteids  in  various  classes  of  animals.  The  myosin 
(v.  Furth)  and  myogen  occur  in  all  classes  of  vertebrates;  the  myogen  is  always 
absent  in  the  invertebrates.  Myoproteid  occurs,  at  least  in  considerable  quantity, 
only  in  fishes.  In  the  muscle  after  cutting  the  nerve  Steyrer  2  found  somewhat 
more  musculin  and  less  myogen  in  the  muscle  juice  than  in  the  normal  muscle. 

Muscle-pigments.  There  is  no  question  that  the  red  color  of  the  muscles 
even  when  completely  freed  from  blood  depends  in  part  on  haemoglobin. 
K.  Morner  has  shown  that  muscle  haemoglobin  is  not  quite  identical  with 
blood-haemoglobin.  The  statement  of  MacMunn,  that  in  the  muscles 
another  pigment  occurs  which  is  allied  to  hsemochromogen  and  called  myo- 
hcematin  by  him,  has  not  been  substantiated,  at  least  for  muscles  of  higher 
animals  (Levy  and  Morner  3).  MacMunn  claims  that  myohaematin  occurs 
in  the  muscles  of  insects,  which  do  not  contain  any  haemoglobin. 

The  reddish-yellow  coloring-matter  of  the  muscles  of  the  salmon  has  been  little 
studied.     Among  the  enzymes  of  the  muscles  besides  traces  of  fibrin  ferment, 

1  For  these  reasons  the  author  is  not  sure  whether  he  has  understood  and  correctly 
given  the  work  of  the  different  investigators. 

2  Przibram,  Hofmeister's  Beitr-igc,  2;  Steyrer,  ibid.,  4. 

3  See  MacMunn,  Phil.  Trans,  of  Roy.  Soc,  177,  part  1,  Journ.  of  Physiol.,  8,  and 
Zeitschr;  f.  physiol.  Chem.,  13;  Levy,  ibid.,  13;  K.  Morner,  Nord.  Med.  Archiv.  Fest- 
band,  1897,  and  Maly's  Jahresber.,  27. 


EXTRACTIVE  BODIES  OF   THE  MUSCLES.  383 

myosin  ferment,  and  amylolytic  ferment  we  must  especially  mention  a  glycolytic 
enzyme  (Brunton    ml  Rhodes)  and  the  proteolytic  enzyme,  closely  studied1  by 

lliniN' mid  Rowland,1  which  maybe  active  in  acid  as  well  as  neutral  and  alkaline 
solutions.    The  power  of  the  muscle  juice  of  destroying  sugar  in  the  presence  <>f 

the  pancreas  (see  Chapter  VIII),  as  first  shown  by  CoHNHEIM,  also  belongs  perhaps 
to  the  enzyme  action. 

Extractive  Bodies  of  the  Muscles. 

The  nitrogenous  extractives  consist  chiefly  of  creatine,  on  an  average  of 
1-4  p.  in.,  in  the  fresh  muscles  containing  water,  also  the  purin bases,  hypoxan- 
thine  and  xanthine,  besides  guanine  and  carnine,  but  chiefly  hypoxanthine. 
The  purin  bases  probably  do  not  occur  as  such  but  as  complex  combinations. 
The  quantity  of  nitrogen  as  purin  bases  amounts,  according  to  Btjrian  and 
Hall,  in  the  fresh  flesh  of  the  horse,  ox,  and  calf  to  0.55,  0.G3,  and  0.71 
p.  m.  respectively,  or  1.3-1.7  p.  m.,  calculated  as  hypoxanthine.  In  the 
embryonic  ox-muscles  KosSEL2  found  more  guanine  than  hypoxanthine. 

Among  the  habitually  occurring  nitrogenous  extractives  we  should  men- 
tion phosphocarnic  acid  and  also  inosinic  acid,  which  is  perhaps  allied  to  it 
and  carnosin. 

Among  the  extractive  subs' ances  is  also  found  the  acid  noliced  by  Limpricht 
in  the  Mesh  of  certain  cyprinidea,  namely,  the  nitrogenized  protic  acid  and  tsocrea- 
tinine  '  found  by  J.  Thesen  in  fish-flesh.  Uric  acid,  urea,  taurin,  and  leucin  are 
found  as  traces  in  the  muscles,  in  certain  cases  only,  of  a  few  species  of  animals. 
In  regard  to  the  amount  of  these  different  extract' ves  in  the  muscles,  Kbuken- 
BERG  and  WAGNER  4  have  shown  that  it  varies  greatly  in  different  animals.  A 
large  quantity  of  urea  is  found  in  the  muscles  of  the  shark  and  ray;  uric  acid  is 
found  in  alligators;  taurin  in  cephalopoda;  glycocoll  in  mollusks,  pecten  irradians; 
and  creatinine  in  luvarus  imperialis,  etc.,  etc.  The  reports  are  very  contra- 
dictory in  regard  to  the  occurrence  of  urea  in  the  muscles  of  higher  animals. 
According  to  the  investigations  of  Kaufmann  and  Schondorff,  confirmed  by 
Brunton-Blakie,1  urea  is  a  regular  constituent  of  the  muscles,  although 
M.  Nencki  and  Kowarski  dispute  this. 

The  xanthine  bodies  with  the  exception  of  carnine  have  been  treated 
on  pages  132-137,  and  therefore  among  the  extractive  bodies  we  will  first 
consider  the  creatine. 

/NH2 
Creatine,  C4H9N302,  (HN)CY  ,   or  methvlguanidin- 

xN(CH3).CH2.COOH 
acetic    acid,  occurs    in    the   muscles    of    vertebrate    animals    in  variable 

'Brunton  and  Rhodes,  Centralbl.  f.  Physiol.,  12;  Hedin  and  Rowland,  Zeitschr. 
f.  physiol.  Chem.,  32. 

2  Burian  and  Hall,  Zeitschr.  f.  physiol.  Chem.,  38;  Kossel,  ibid.,  S,  408. 

8  See  Limpricht,  Annal.  de  Chem.  u.  Pharm.,  127,  and  Thesen,  Zeitschr.  f.  physiol. 
Chem.,  24. 

4  Zeitschr.  f.  Biologie,  21. 

5  Kaufmann,  Arch,  de  Physiol.  (5),  6;  Schondorff,  Pfliiger's  Arch.,  (52;  Xencki 
and  Kowarski,  Arch.  f.  exp.  Path.  u.  Pharm.,  36;  Brunton-Blakie,  Journ.  of  Physiol., 
23,  Supplement. 


3S4  MUSCLE. 

amounts  in  different  species;  the  largest  quantity  is  found  in  birds. 
According  to  Monari  the  amount  is  increased  by  work  when  a  part  of 
the  creatine  is  transformed  into  creatinine.  It  is  also  found  in  the 
brain,  blood,  transudates,  and  the  amniotic  fluid.  Creatine  may  be  pre- 
pared synthetically  from  cyanamide  and  sarcosin  (methylglycocoll) .  On 
boiling  with  baryta-water  it  decomposes  with  the  addition  of  water  and 
yields  urea,  sarcosin,  and  certain  other  products.  Because  of  this  be- 
havior several  investigators  consider  creatine  as  a  step  in  the  formation  of 
urea  in  the  organism.  On  boiling  with  acids  creatine  is  easily  converted, 
with  the  elimination  of  water,  into  creatinine,  C4H7N30,  which  occurs  in 
urine,  and  which  has  also  been  found  in  the  muscles  of  the  dog  by  Monari  * 
(see  Chapter  XV). 

Creatine  crystallizes  in  hard,  colorless,  monoclinic  prisms  which  lose 
their  water  of  crystallization  at  100°  C.  It  is  soluble  in  74  parts  of  water 
at  the  ordinary  temperature  and  9410  parts  absolute  alcohol.  It  dissolves 
more  easily  with  the  aid  of  heat.  Its  watery  solution  has  a  neutral  reaction. 
Creatine  is  not  dissolved  by  ether.  If  a  creatine  solution  is  boiled  with 
precipitated  mercuric  oxide,  this  is  reduced,  especially  in  the  presence  of 
alkali,  to  mercury  and  oxalic  acid,  and  the  foul-smelling  methyluramine 
(methylguanidine)  is  developed.  A  solution  of  creatine  in  water  is  not  pre- 
cipitated by  basic  lead  acetate,  but  gives  a  white,  flaky  precipitate  with 
mercurous  nitrate  if  the  acid  reaction  is  neutralized.  When  boiled  for  an 
hour  with  dilute  hydrochloric  acid  creatine  is  converted  into  creatinine  and 
may  be  identified  by  its  reactions.  On  boiling  with  formaldehyde  it  can 
be  transformed  into  dioxymethylencreatinine,  which  crystallizes  readily 
(Jaffe  2). 

The  preparation  and  detection  of  creatine  is  best  performed  by  the 
following  method  of  Neubauer,3  which  was  first  used  in  the  preparation 
of  creatine  from  muscles :  Finely  cut  meat  is  extracted  with  an  equal  weight 
of  water  at  50°  to  55°  C.  for  10-15  minutes,  pressed  and  extracted  again 
with  water.  The  proteids  are  removed  from  the  united  extracts  as  far  as 
possible  by  coagulation  at  boiling  heat,  the  filtrate  precipitated  by  the  care- 
ful addition  of  basic  lead  acetate,  the  lead  removed  from  this  filtrate  by  H2S 
and  carefully  concentrated  to  a  small  volume.  The  creatine,  which  crys- 
tallizes in  a  few  days,  is  collected  on  a  filter,  washed  with  alcohol  of  88  per 
cent,  and  purified  when  necessary,  by  recrystallization.  The  quantitative 
estimation  of  creatine  is  performed  according  to  the  same  method. 

Isocreatinine  is  a  creatinine  isomeric  with  ordinary  creatinine  and  found  by 
Thesrn  4  in  the  flesh  of  the  codfish.  It  crystallizes  in  yellow  needles  or  plates, 
is  more  soluble  in  cold  water,  but  more  insoluble  in  alcohol,  than  the  ordinary 
creatinine,  and  gives  a  picrate  which  is  readily  soluble  and  a  zinc  chloride  com- 

1Maly's  Jahresber.,  19,  296. 

2  Ber.  d.  d.  chem.  Gesellsch.,  35. 

3  Zeitschr.  f.  physiol.  Chem.,  2  and  6. 
*  L.  c. 


CARNINE  AND  PIIOSPIIOCARNIC  ACID.  385 

bination  which  is  relatively  readily  soluble.     It  gives  Wkvi, 's  reaction  less  rapidly, 
and  does  not  give  methylguanidine  on  treatment  with  potassium  permanganate. 

Carnine,  C8H8N403  +  H20,  is  one  of  the  substances  found  by  Weidel  in 
American  meat  extract.  It  has  also  been  found  by  Krukenberg  and 
Wagner  in  frog-muscles  and  in  the  flesh  of  fishes,  and  by  Pouchet  '  in  the 
urine.     Carnine  may  be  transformed  into  hypoxanthine  by  oxidation. 

Carnine  has  been  obtained  as  a  white  crystalline  mass.  It  dissolves 
with  difficulty  in  cold  water,  but  more  readily  in  warm.  It  is  insoluble  in 
alcohol  and  ether.  It  dissolves  in  warm  hydrochloric  acid  and  yields  a  salt 
crystallizing  in  shining  needles,  which  gives  a  double  combination  with 
platinum  chloride.  Its  watery  solution  is  precipitated  by  silver  nitrate,  but 
this  precipitate  is  dissolved  neither  by  ammonia  nor  by  warm  nitric  acid. 
Carnine  does  not  give  the  so-called  Weidel 's  xanthine  reaction.  Its 
watery  solution  is  precipitated  by  basic  lead  acetate;  still  the  lead  com- 
bination may  be  dissolved  on  boiling. 

Carnine  is  prepared  by  the  following  method:  The  meat  extract  diluted 
with  water  is  completely  precipitated  by  baryta-water.  The  filtrate  is 
precipitated  by  basic  lead  acetate,  the  lead  precipitate  boiled  with  water, 
filtered  while  hot,  and  sulphuretted  hydrogen  passed  through  the  filtrate. 
Remove  the  lead  sulphide  from  the  filtrate  and  concentrate  strongly.  The 
concentrated  solution  is  now  completely  precipitated  with  silver  nitrate,  the 
precipitate  washed  free  from  silver  chloride  by  ammonia,  and  the  carnine 
silver  oxide  suspended  in  water  and  treated  with  sulphuretted  hydrogen. 

Carnosin,  C„H,4X403,  has  been  isolated  by  Gulewttsch  and  Aomiradzibi  2 
from  meat  extracts.  It  is  a  base  which  is,  perhaps,  related  to  arginin,  and  is 
readily  soluble  in  water,  crystallizing  in  flat  needles.  It  is  precipitated  by 
phosphotungstic  acid  and  by  silver  nitrate  in  the  presence  of  an  excess  of  barium 
hydrate  and  forms  a  copper  compound  which  crystallizes  in  hexagonal  plates. 

The  base,  musculamine,  isolated  by  Etakd  and  Vila,  is,  according  to  Poster- 
NAK,a  nothing  but  cadaverin. 

Phosphocarnic  acid  4  is  a  complicated  substance,  first  isolated  by  Siegfried 
from  meat  extracts,  which  yields  as  cleavage  products  succinic  acid,  paralactic 
acid,  carbon  dioxide,  phosphoric  acid,  and  a  carbohydrate  group,  besides  the 
previously  mentioned  carnic  acid,  which  is  identical  with,  or  nearly  related  to 
antipeptone.  It  stands,  according  to  Siegfried,  in  close  relationship  to  the 
nucleins,  and  as  it  yields  peptone  (carnic  acid),  it  is  designated  as  a  nuclcon  by 
Siegfried.  Phosphocarnic  acid  may  be  precipitated  as  an  iron  combination, 
cartiijrrri/i,  from  the  extract  of  the  muscles  free  from  proteids.  The  quantity  of 
phosphocarnic  acid,  calculated  as  carnic  acid,  can  be  determined  by  multiplying 

1  Weidel,  Annal.  d.  Chem.  u.  Pharm.,  15S;  Wagner,  Sitzungsber.  d.  Wurab.  phys.- 
med.  Gesellsch.,  1S83;  Pouchet,  cited  from  Xeubauer-Huppert,  Analyse  des  Harnes, 
10.  Aufl.,  335. 

:  Zeitschr.  f.  physiol.  Chem.,  30. 

3  Etard  and  Vila,  Compt.  rend.,  13o;   Posternak,  ibid. 

*  In  regard  to  carnic  acid  and  phosphocarnic  acid,  see  the  works  of  Siegfried, 
Du  Bois-Reymond's  Arch.,  1S94,  Ber.  d.  deutseh.  Chem.  Gesellsch..  2s,  and  Zeitschr. 
f.  physiol.  Chem..  21  and  2s ;  M.  Midler,  ibid.,  22;  Kruger,  ibid.,  22  and  28;  Balke  and 
Ide,  ibid.,  21,  and  Balke,  ibid.,  22;   Macleod,  ibid.,  28. 


386  MUSCLE. 

the  quantity  of  nitrogen  in  the  combination  by  the  factor  6.1237  (Balke  and 
Ide).  In  this  way  Siegfried  found  0.57-2.4  p.  m.  carnic  acid  in  the  resting 
muscles  of  the  dog,  and  M.  Muller  1-2  p.  m.  in  the  muscle  of  adults  and  a  maxi- 
mum of  0.57  p.  m  in  those  of  new-born  infants.  Phosphocarnic  acid  has  not 
been  prepared  in  the  pure  state  and  possesses  on  this  account  a  variable  compo- 
sition; according  to  Siegfried,  it  serves  as  a  source  of  energy  in  the  muscles  and 
is  consumed  during  work.  Besides,  by  means  of  its  property  of  forming  soluble 
salts  with  the  alkaline  earths,  as  also  an  iron  combination  soluble  in  alkalies,  it 
acts  as  a  means  of  transportation  for  these  bodies  in  the  animal  body. 

Phosphocarnic  acid  is  prepared  from  the  extract  free  from  proteid  by  first 
removing  the  phosphate  by  CaCl2  and  NH3.  The  acid  is  precipitated  by  ferric 
chloride  from  the  nitrate  while  boiling,  as  carniferrin. 

Inosinic  acid  has  been  discussed  on  page  128.  We  must  also  include  among: 
the  nitrogenous  extractives  those  bodies  which  were  first  discovered  by  Gautier  1 
and  which  occur  only  in  very  small  quantities,  namely,  the  leucomaines,  xantho- 
creatinine,  C5H10N4O,  crusocreatinine,  C5H8N40,  amphicreatinine,  C0H19N7O4,  and 
pseadoxanthine,  C4H5N50. 

In  the  analysis  of  meat  and  for  the  detection  and  separation  of  the  various- 
extractive  bodies  of  the  same  we  make  use  of  the  systematic  method  as  suggested 
by  Gautier,2  for  details  of  which  the  reader  is  referred  to  the  original  article. 

The  non-nitrogenous  extractive  bodies  of  the  muscles  are  inosite,  glyco- 
gen, sugar,  and  lactic  acid. 

Inosite,  C6H1206  +  H20  =  C6Ha(OH)e  +  H20.  This  body,  discovered  by 
Scherer,  is  not  a  carbohydrate,  but  a  hexahydroxybenzene  (Maqtjenne  3). 
With  hydriodic  acid  it  yields  benzene  and  tri-iodophenol.  Inosite  is  found 
in  the  muscles,  liver,  spleen,  leucocytes,  kidneys,  suprarenal  capsule,  lungs, 
brain,  testicles,  and  in  the  urine  in  pathological  cases,  and  as  traces  in 
normal  urine.  It  is  found  very  widely  distributed  in  the  vegetable  king- 
dom, especially  in  unripe  fruits,  and  in  green  beans  (phaseolus  vulgaris), 
and  therefore  it  is  also  called  phaseomannit.  According  to  Winterstein 
a  phosphorized  compound  occurs  in  the  vegetable  kingdom  which  yields 
inosite  as  a  cleavage  product.  This  compound  is,  according  to  Posternak,4 
probably  oxymethylphosphoric  acid,  which  also  yields  inosite  on  decom- 
position by  condensation. 

Inosite  crystallizes  in  large,  colorless,  rhombic  crystals  of  the  monoclinic 
system,  or,  if  not  pure  and  if  only  a  small  quantity  crystallizes,  it  forms 
groups  of  fine  crystals  similar  to  cauliflower.  It  loses  its  water  of  crystalliza- 
tion at  110°  C,  also  if  exposed  to  the  air  for  a  long  time.  Such  exposed 
<t  tals  are  non-transparent  and  milk-white.  The  crystals  melt  at  225°  C. 
when  dry.  Inosite  dissolves  in  7.5  parts  of  water  at  ordinary  temperature, 
and  the  solution  has  a  sweetish  taste.  It  is  insoluble  in  strong  alcohol  and 
i.i  ether.     It  dissolves  cupric  hydrate  in  alkaline  solutions,  but  does  not 

See  Mary's  Jahresber.,  10,  523. 

2  Ibid.,  22,  355. 

3  Bull,  (le  la  Soc.  Chim.  (2),  47  and  48;  Compt.  rend.,  104. 

MVinterstein,  Ber.  d.  d.  chem.  Gesellsch..  30;  Posternak,  Contribution  a  1 'etude 
chim  de  1 'assimilation  chlorophyllienne,  Revue  generate  de  Botanique,  12  (1900). 


INOSITi:.  387 

reduce  on  boiling.  It  gives  negative  results  with  Moore's  test  and  with 
Bottger-Almen's  bismuth  test.     It   does  not  fennent  with  beer-yeast, 

but  may  undergo  lactic-  and  butyric-acid  fermentation.  The  lactic  acid 
formed  thereby  Is  sarcolactic  acid  according  to  HlLOER,  and  fermenta- 
tion lactic  acid  according  to  Vohl.1  Inosite  is  oxidized  into  rhodizonic 
acid  by  an  excess  of  nitric  acid,  and  the  following  reactions  depend  upon 
this  behavior: 

If  inosite  is  evaporated  to  dryness  on  platinum-foil  with  nitric  acid  and 
the  residue  treated  with  ammonia  and  a  drop  of  calcium-chloride  solution 
and  carefully  re-evaporated  to  dryness,  a  beautiful  rose-red  residue  is 
obtained  (Sciierer's  inosite  test).  If  we  evaporate  an  inosite  solution  to 
incipient  dryness  and  moisten  the  residue  with  a  little  mercuric-nitrate 
solution,  there  is  obtained  a  yellowish  residue  on  drying,  which  becomes  a 
beautiful  red  on  strongly  heating.  The  coloration  disappears  on  cooling, 
but  it  reappears  on  gently  warming  (Gallois'  inosite  test). 

To  prepare  inosite  from  a  liquid  or  from  a  watery  extract  of  a  tissue, 
the  proteids  are  first  removed  by  coagulation  at  boiling  heat.  The  filtrate 
is  precipitated  by  sugar  of  lead,  this  filtrate  boiled  with  basic  lead  acetate 
and  allowed  to  stand  24-48  hours.  The  precipitate  thus  obtained,  which 
contains  all  the  inosite,  is  decomposed  in  water  by  H28.  The  filtrate  Is 
strongly  concentrated,  treated  with  2-4  vols,  hot  alcohol,  and  the  liquid 
removed  as  soon  as  possible  from  the  tough  or  flaky  masses  which  ordinarily 
separate.  If  no  crystals  separate  from  the  liquid  within  twenty-four  hours, 
then  treat  with  ether  until  the  liquid  has  a  milky  appearance  and  allow  it 
to  stand.  In  the  presence  of  a  sufficient  quantity  of  ether,  crystals  of 
inosite  separate  within  twenty-four  hours.  The  crystals  thus  obtained, 
a-  also  those  which  are  obtained  from  the  alcoholic  solution  directly,  are 
recrystallized  by  redissolving  in  very  little  boiling  water  and  the  addition 
of  2^4  vols,  of  alcohol. 

Glycogen  is  a  constant  constituent  of  the  living  muscle,  while  it  may  be 
absent  in  the  dead  muscle.  The  quantity  of  glycogen  varies  in  the  different 
muscles  of  the  same  animal.  Bohm  2  found  10  p.  m.  glycogen  in  the  muscles 
of  cats,  and  moreover  he  found  a  greater  amount  in  the  muscles  of  the 
extremities  than  in  those  of  the  rump.  Schondorff  has  found  a  maxi- 
mum of  37.2  p.  m.  in  the  dog  muscle.  The  statements  as  to  the  quantity 
of  glycogen  in  the  heart  differ  somewhat;  although  the  heart  is  considered 
as  somewhat  poorer  in  glycogen  than  the  other  muscles,  still  this  difference 
is  not  very  great  and  can  be  explained  by  the  ready  disappearance  of 
glycogen  from  the  heart  after  death,  as  well  as  after  starvation  and  after 
strong  work  (Boruttau,  Jensen  3).  Work  and  the  food  have  a  great 
influence  upon  the  quantity  of  glycogen.     Bohm  found  1-4  p.  m.  glycogen 

1  Hilger,  Annal.  d.  Chera.  u.  Pharm.,  160;  Vohl,  Ber.  d.  d.  chem.  Gesellsch.,  9. 

2  Bohm,  Pfliiger's  Arch.,  23,  44;   Schondorff,  ibid.,  99. 

3  Boruttau,  Zeitschr.  f.  physiol.  Chem.,  18;   Jensen,  ibid.,  35. 


388  MUSCLE. 

in  the  muscles  of  fasting  animals,  and  7-10  p.  m.  after  partaking  of  food. 
As  stated  in  Chapter  VIII,  work,  starvation,  and  lack  of  carbohydrates 
in  the  food  cause  the  glycogen  to  disappear  earlier  from  the  liver  than  from 
the  muscles. 

The  sugar  of  the  muscles,  of  which  traces  only  occur  in  the  living  muscle, 
and  which  is  probably  formed  after  the  death  of  the  muscle  from  the  muscle- 
glycogen,  is,  according  to  the  investigations  of  Panormoff,  in  part  dex- 
trose, but  consists  chiefly  of  maltose  (Osborne  and  Zobel  *)  with  some 
dextrin. 

Lactic  Acids.  Of  the  oxypropionic  acids  with  the  formula  C3H603 
there  is  one,  ethylene  lactic  acid,  CH2(OH).CH2.COOH,  which  is  not  found 
in  the  animal  body  and  therefore  has  no  physiological  chemical  interest. 

CH3 

Indeed  only  a-oxypropionic  acid  or  ethylidene  lactic  acid,  CH(OH),  of 

COOH 
which  there  are  three  physical  isomers,  is  of  importance.  These  three 
ethylidene  lactic  acids  are  the  ordinary,  optically  inactive  fermentation 
lactic  acid,  the  dextrorotatory  paralactic  or  sarcolactic  acid,  and 
the  LuEvolactic  acid  obtained  by  Schardinger  by  the  fermentation  of 
cane-sugar  by  means  of  a  special  bacillus.  This  lsevolactic  acid,  which 
has  also  been  detected  by  Blachstein  in  the  culture  of  Gaffky  's  typhoid 
bacillus  in  a  solution  of  sugar  and  peptone,  and  which  is  formed  by  vari- 
ous vibriones,  need  not  be  described  here.2 

The  fermentation  lactic  acid,  which  is  formed  from  lactose  by  allow- 
ing milk  to  sour  and  by  the  acid  fermentation  of  other  carbohydrates, 
is  considered  to  exist  in  small  quantities  in  the  muscles  (Heintz),  in  the 
gray  matter  of  the  brain  (Gscheidlen  3),  and  in  diabetic  urine.  During 
digestion  this  acid  is  also  found  in  the  contents  of  the  stomach  and  intes- 
tine, and  as  alkali  lactate  in  the  chyle.  The  paralactic  acid  is,  at  all 
events,  the  true  acid  of  meat  extracts,  and  this  alone  has  been  found  with 
certainty  in  dead  muscle.  The  lactic  acid  which  is  found  in  the  spleen, 
lymphatic  glands,  thymus,  thyroid  gland,  blood,  bile,  pathological  trans- 
udates, osteomalacious  bones,  in  perspiration,  in  puerperal  fever,  and  in 
the  urine  after  fatiguing  marches,  in  acute  yellow  atrophy  of  the  liver,  in 
poisoning  by  phosphorus,  and  especially  after  extirpation  of  the  liver 
seems  to  be  paralactic  acid. 

1  Panormoff,  Zeitschr.  f.  physiol.  Chem.,  17 j  Osborne  and  Zobel,  Journ.  of 
Physiol.,  29. 

2  See  Schardinger,  Monatshefte  f.  Chem.,  11;  Blachstein,  Arch,  des  sciences  biol. 
de  St.  PStersbourg,  1,  199;  Kuprianow,  Arch.  f.  Hygiene,  19;  and  Gosio,  ibid.,  21. 

8  Heintz,  Annal.  d.  Chem.  u.  Pharm.,  157,  and  Gscheidlen,  Pfliiger's  Arch.,  8, 
171. 


LACTIC  ACIDS.  389 

The  origin  of  paralactic  acid  in  the  animal  organism  has  been  sought 
by  several  investigators,  who  took  for  basis  the  researches  of  Gaglio, 
Minkowski,  and  Araki,  in  a  decomposition  of  proteid  in  the  tissues. 
Gaglio  claims  jsl  lactic-acid  formation  by  passing  blood  through  the  kid- 
neys and  lungs.  He  also  found  0.3-0.5  p.  m.  lactic  acid  in  the  blood  of 
a  dog  after  proteid  food,  and  only  0.17-0.21  p.  m.  after  fasting  for  forty- 
eight  hours.  According  to  Minkowski  the  quantity  of  lactic  acid  elimi- 
nated by  the  urine  in  animals  with  extirpated  livers  is  increased  with  pro- 
teid food,  while  the  administration  of  carbohydrates  has  no  effect.  Araki 
has  also  shown  that  if  we  produce  a  scarcity  of  oxygen  in  animals  (dogs, 
rabbits,  and  hens)  by  poisoning  with  carbon  monoxide,  by  the  inhalation 
of  air  deficient  in  oxygen,  or  by  any  other  means,  a  considerable  elimina- 
tion of  lactic  acid  (besides  dextrose  and  also  often  albumin)  takes  place 
through  the  urine,  an  observation  which  has  been  confirmed  by  Saito 
and  Katsuyama.1  As  a  scarcity  of  oxygen,  according  to  the  ordinary 
statements,  produces  an  increase  of  the  proteid  katabolism  in  the 
body,  the  increased  elimination  of  lactic  acid  in  these  cases  must  be 
due  in  part  to  an  increased  proteid  destruction  and  in  part  to  a  dimin- 
ished oxidation. 

Araki  has  not  drawn  such  a  conclusion  from  his  experiments,  but 
he  considers  the  abundant  formation  of  lactic  acid  to  be  due  to  a  cleavage 
of  the  sugar  formed  from  the  glycogen.  He  found  that  in  all  cases  where 
lactic  acid  and  sugar  appeared  in  the  urine  the  quantity  of  glycogen  in 
the  liver  and  muscles  was  always  diminished.  He  also  calls  attention  to 
the  fact  that  dextrolactic  acid  may  be  formed  from  glycogen,  as  directly 
observed  by  Ekunina,2  and  also  to  the  numerous  observations  on  the 
formation  of  lactic  acid  and  the  consumption  of  glycogen  in  muscular 
activity.  "Without  denying  the  possibility  of  a  formation  of  lactic  acid 
from  proteid,  he  states  that  with  lack  of  oxygen  we  have  to  deal  with  an 
incomplete  combustion  of  the  lactic  acid  derived  by  a  cleavage  of  the  sugar. 
Hoppe-Seyler  3  also  positively  defends  the  view  as  to  the  formation  of 
lactic  acid  from  carbohydrates.  He  was  of  the  view  that  lactic  acid  is 
produced  from  the  carbohydrates  by  the  cleavage  of  the  sugar  only  with 
lack  of  oxygen,  while  with  sufficient  oxygen  the  sugar  is  burned  into  carbon 
dioxide  and  water.  The  formation  of  lactic  acid  in  the  absence  of  free 
oxygen  and  in  the  presence  of  glycogen  or  dextrose  is,  according  to  Hopim:- 
Seyler,  very  probably  a  function  of  all  living  protoplasm.  There  is 
no  direct  proof  for  such  a  view.    In  the  anaerobic  metabolism  of  the  animal 

1  Gaglio,  Du  Bois-Reymond 's  Arch.,  1886;  Minkowski,  Arch.  exp.  Path.  u.  Pharm., 
21  and  31 ;  Araki,  Zeitschr.  f.  physiol.  Chem.,  15,  16,  1",  and  19;  Saito  and  Katsuyama, 
ibid.,  32. 

2  Journ.  f.  prakt.  Chem.  (X.  F.),  21. 

5  Virchow's  Festschrift,  also  Ber.  d.  deutsch.  chem.  Gesellsch.,  25,  Referatb.,  685. 


390  MUSCLE. 

cells  carbon  dioxide  and  alcohol  are  formed  from  the  sugar  according  to 
the  investigations  of  Simacek;  l  and  when  the  cells,  as  Stoklasa  and  his 
collaborators  have  shown,  contain  a  lactic-acid-forming  enzyme,  it  is 
not  known  what  kind  of  lactic  acid  is  here  produced.  According  to 
Morishima,  an  increase  in  the  lactic  acid  in  the  liver  occurs  after  death, 
probably  from  the  liver  glycogen,  but  this  acid  is  chiefly  fermentation 
lactic  acid.  Asher  and  Jackson  2  experimented  by  transfusing  blood  (with 
and  without  the  addition  of  sugar)  through  the  lower  extremities  of  dogs, 
and  neither  in  these  experiments  nor  in  those  where  the  larger  organs 
(liver  and  abdominal  viscera)  were  excluded  from  the  circulation  could 
they  detect  any  increase  of  lactic  acid  due  to  the  sugar.  At  present  there 
seems  to  be  a  tendency  towards  the  view  that  the  cause  for  the  increased 
formation  of  lactic  acid  with  lack  of  oxygen  is  to  be  sought  for  in  the  in- 
creased destruction  of  proteids.  PhosphocarrnVacid  is  considered  by  Sieg- 
fried as  another  source  of  sarcolactic  acid. 

The  lactic  acids  are  amorphous.  They  have  the  appearance  of  color- 
less or  faintly  yellowish,  acid-reacting  syrups  which  mix  in  all  proportions 
with  water,  alcohol,  or  ether.  The  salts  are  soluble  in  water,  and  most  of 
them  also  in  alcohol.  The  two  acids  are  differentiated  from  each  other  by 
their  different  optical  properties — paralactic  acid  being  dextrogyrate,  while 
fermentation  lactic  acid  is  optically  inactive — also  by  their  different  solu- 
bilities and  the  different  amounts  of  water  of  crystallization  of  the  calcium 
and  zinc  salts.  The  zinc  salt  of  fermentation  lactic  acid  dissolves  in  5S-63 
parts  of  water  at  14-15°  C,  and  contains  18.18  per  cent  water  of  crystalli- 
zation, corresponding  to  the  formula  Zn(C3H503)2-|-3H20.  The  zinc  salt 
of  paralactic  acid  dissolves  in  17.5  parts  of  water  at  the  above  •  tempera- 
ture and  contains  ordinarily  12.9  per  cent  water,  corresponding  to  the  formula 
Zn(C3H503)2  +  2H20.  The  calcium  salt  of  fermentation  lactic  acid  dis- 
solves in  9.5  parts  water  and  contains  29.22  per  cent  (  =  5  molecules)  water 
of  crystallization,  while  calcium  paralactate  dissolves  in  12.4  parts  water 
and  contains  24.83  or  26.21  per  cent  (  =  4  or  4£  molecules)  water  of  crystalli- 
zation. Both  calcium  salts  crystallize,  not  unlike  tyrosin,  in  spears  or 
tufts  of  very  fine  microscopic  needles.  Hoppe-Seyler  and  Araki,  who 
have  closely  studied  the  optical  properties  of  the  lactic  acids  and 
lactates,  consider  the  lithium  salt  as  best  suited  for  the  preparation  and 
quantitative  estimation  of  the  lactic  acids.  The  lithium  salt  contains 
7.29  per  cent  Li.  For  further  information  as  to  the  salts  and  specific 
rotation  of  the  lactic  acids  see  Hoppe-Seyler,  Thierf elder's  Handbuch, 
7.  Aufl.,  1903. 

Lactic  acids  may  be  detected  in  organs  and  tissues  in  the  following 

1  Simacek,  Centralbl.  f.  Physiol.,  17;  Stoklasa,  Jelinek  and  Cerny,  ibid.,  16. 

2  Morishima,  Arch.  f.  exp.  Path.  u.  Pharnx,  43;  Asher  and  Jackson,  Zeitschr.  f. 
Biologie,  41. 


LACTIC  .\cins,  FAT,  LECITHIN  3    I 

manner:  After  complete  extraction  with  water,  the  proteid  is  removed  by 
coagulation  at  boiling  temperature  and  the  addition  of  a  small  quantity  of 
Bulphuric  acid.    The  liquid  is  then  exactly  neutralized  while  boiling  with 

caustic  baryta,  and  then  evaporated  to  a  syrup  alter  filtration.     The  residue 

is  precipitated  with  absolute  alcohol,  and  the  precipitate* completely  ex- 
tracted with  alcohol.  The  alcohol  is  entirely  distilled  from  the  united  alco- 
holic extracts,  and  the  neutral  residue  is  shaken  with  ether  to  remove  the 

fat.  The  residue  is  dissolved  in  water  and  phosphoric  acid  added,  and  re- 
peatedly shaken  with  fresh  quantities  of  ether,  which  dissolves  the  lactic  acid. 
The  other  is  now  distilled  from  the  several  ethereal  extracts,  the  residue 
dissolved  in  water,  and  this  solution  carefully  warmed  on  the  water-bath  to 
remove  the  last  traces  of  ether  and  volatile  acids.  A  solution  of  zinc  lac- 
tate is  prepared  from  this  filtered  solution  by  boiling  with  zinc  carbonate, 
and  this  is  evaporated  until  crystallization  commences  and  then  allowed  to 
stand  over  sulphuric  acid.  An  analysis  of  the  salts  is  necessary  in  careful 
work.  According  to  Hkffter  '  in  muscles  not  having  undergone  rigor  mortis 
the  lactic  acid  can  be  extracted  more  easily  by  alcohol  than  by  water. 

/'al  is  never  absent  in  the  muscles.  Some  fat  is  always  found  in  the 
intermuscular  connective  tissue;  but  the  muscle-fibres  themselves  also  con- 
tain fat.  The  quantity  of  fat  in  the  real  muscle  substance  is  always  small, 
usually  amounting  to  about  10  p.  m.  or.  somewhat  more.  A  considerable 
quantity  of  fat  in  the  muscle-fibres  is  only  found  in  fatty  degeneration.  A 
part  of  the  muscle-fat  can  be  readily  extracted,  while  another  part  can  be 
extracted  only  with  the  greatest  difficulty.  This  latter  part,  it  is  claimed, 
exists  finely  divided  in  the  contractile  substance  itself  and  is  richer  in  free 
fatty  acids,  standing,  according  to  Zuntz  and  Bogdanow,2  in  close  rela- 
tionship to  the  activity  of  the  muscles  because  it  is  consumed  during 
work.  Lecithin  is  a  regular  constituent  of  the  muscles,  and  it  is  quite 
possible  that  the  fat  which  is  difficult  of  extraction  and  which  is  rich  in 
fatty  acids  depends  in  part  on  a  decomposition  of  the  lecithin. 

The  Mineral  Bodies  of  the  Muscles.  The  ash  remaining  after  burning 
the  muscle,  which  amounts  to  about  10-15  p.  m.,  calculated  on  the  moist 
muscle,  is  acid  in  reaction.  The  largest  constituents  are  potassium  and 
phosphoric  acid.  Next  in  amount  we  have  sodium  and  magnesium,  and 
lastly  calcium,  chlorine,  and  iron  oxide.  Sulphates  exist  only  as  traces  in 
the  muscles,  but  are  formed  by  the  burning  of  the  proteids  of  the  muscles, 
and  therefore  occur  in  abundant  quantities  in  the  ash.  The  muscles  con- 
tain such  a  large  quantity  of  potassium  and  phosphoric  acid  that  potassium 
phosphate  seems  to  be  unquestionably  the  predominating  salt.  Chlorine  is 
found  in  such  insignificant  quantities  that  it  is  perhaps  derived  from  a  con- 
tamination with  blood  or  lymph.  The  quantity  of  magnesium  is,  as  a  rule, 
considerably  greater  than  that  of  calcium.  Iron  occurs  only  in  very  small 
amounts.     Schmey  3  found  variations  between  0.0129  p.  m.  (rabbits)  and 

1  Arch.  f.  exp.  Path.  u.  Pharra.,  38. 

2  Du  Boirf-Revmond's  Arch.,  1897. 
sZeitschr.  i.  physiol.  Chem.,  39. 


392  MUSCLE. 

0.0793  p.  m.  (human),  calculated  on  the  fresh  muscle  substance.  The  heart- 
muscle  was  comparatively  richer  in  iron,  0.06-0.109  p.  m. 

The  importance  of  the  various  mineral  bodies  for  the  function  of  the 
muscles  has  been  studied  by  several  experimenters  (Loeb,  Lingle,  Howell, 
Overton,  Langendorff  and  Huek,  and  others  *).  Further  proof  as  to  the 
previously  discussed  ion  action  of  the  electrolytes  and  the  antagonism  of 
various  ions  has  been  given  by  many  very  interesting  investigations.  These 
researches  also  indicate  that  each  of  the  ions  Na,  Ca,  and  K  plays  a  certain 
part  in  the  maintenance  of  the  excitability,  in  the  contraction  and  in  the 
fatigue  of  the  muscle  (heart) ;  still  these  investigations  have  not  led  to  con- 
cordant results,  so  that  we  are  not  yet  clear  on  the  action  of  these  ions. 
Nevertheless  it  seems  to  be  established  that  the  combined  action  of  various 
ions  is  a  necessity  for  the  normal  function  of  the  muscles.  It  has  also 
been  shown  that  it  is  possible  to  maintain  the  muscle  (the  heart)  in  regular 
activity  for  a  long  time  by  means  of  a  transfusion  of  liquid  saturated 
with  oxygen  and  which  contained  about  7  p.  m.  NaCl,  besides  small 
amounts  of  CaCl2  (0.2  p.  m.),  KC1  (0.1  p.  m.),  and  NaHC03  (0.1  p.  m.). 

The  gases  of  the  muscles  consist  of  large  quantities  of  carbon  dioxide 
besides  traces  of  nitrogen. 

In  regard  to  the  permeability  of  the  muscles  for  various  bodies  there  are 
the  complete  investigations  of  Overton.2  The  different  sheaths  of  the 
muscles,  the  sarcolemma  and  perimysium  internum,  offer  no  very  great 
resistance  to  the  diffusion  of  most  soluble  crystalloid  compounds,  while 
the  muscle-fibres,  on  the  contrary  (exclusive  of  the  sarcolemma) ,  are  almost 
if  not  entirely  impervious  to  most  inorganic  compounds  and  for  many  organic 
compounds.  The  muscle-fibres  themselves  are  actually  semipermeable  struc- 
tures which  are  permeable  for  water  but  not  for  the  molecules  or  ions  of 
sodium  chloride  and  of  potassium  phosphate.  The  muscle-fibres,  as  well  as 
the  various  sheaths,  are  impermeable  to  colloids. 

The  behavior  of  the  numerous  bodies  investigated  cannot  be  discussed 
in  this  work.  The  general  rule  is  as  follows :  All  compounds  which,  besides 
a  marked  solubility  in  water,  are  readily  soluble  in  ethyl  ether,  in  the 
higher  alcohols,  in  olive-oil  and  in  similar  organic  solvents,  or  are  not  much 
less  soluble  in  the  last-mentioned  solvents  than  in  water,  pass  through  the 
living  muscle-fibres  (as  in  animal-  and  plant-cells)  with  great  ease.  The 
greater  the  difference  between  the  solubility  of  a  compound  in  water  and 
in  the  other  solvents  mentioned,  the  slower  does  the  passage  into  the 
muscle-fibres  take  place.  The  permeability  changes  essentially  on  the 
death  of  the  muscle. 

1  Loeb,  Amer.  Journ.  of  Physiol.,  .1,  and  Pfliiger's  Arch.,  91;  Lingle,  Amer.  Journ. 
of  Physiol.,  4  (also  references  to  literature);  Overton,  Pfliiger's  Arch.,  92;  Langendorff 
and  Huek,  ibid.,  96. 

2  Pfliiger's  Arch.,  92. 


RIGOR  MORTIS.  393 

The  living  muscle-fibres  are  readily  permeable  to  oxygen,  carbon  dioxide, 
and  ammonia,  while  the  hexoses  and  disaccharides  do  not  readily  pass 
into  them.  It  is  very  remarkable  that  a  great  portion  of  those  compounds 
which  take  part  in  the  normal  metabolism  of  plants  and  animals  belong  to 
those  bodies  to  which  the  muscle-fibres  (and  also  other  cells)  are  entirely  or 
at  least  nearly  impermeable.  On  the  contrary,  derivatives  can  be  pre- 
pared from  these  bodies  which  pass  into  the  cells  very  readily,  and  <  >\  i:k- 
ton  finds  that  it  is  not  impossible  that  the  organism  in  part  makes  use  of 
a  similar  artifice  in  order  to  regulate  the  concentration  of  the  nutritive 
bodies  within  the  protoplasm. 

Rigor  Mortis  of  the  Muscles.  If  the  influence  of  the  circulating  oxygen- 
ated  blood  is  removed  from  the  muscles,  as  after  the  death  of  the  animal  or 
by  ligature  of  the  aorta  or  the  muscle-arteries  (Stkxson's  test),  rigor  mortis 
sooner  or  later  takes  place.  The  ordinary  rigor  appearing  under  these 
circumstances  is  called  the  spontaneous  or  the  fermentative  rigor,  because  it 
seems  to  depend  in  part  on  the  action  of  an  enzyme.  A  muscle  may  also 
become  stiff  for  other  reasons.  The  muscles  may  become  momentarily 
stiff  by  warming,  in  the  case  of  frogs,  to  40°,  in  mammalia,  to  48-50°,  and 
in  birds,  to  53°  C.  The  heat-rigor  depends  upon  the  coagulation  of  certain 
proteids,  and  its  occurrence  at  lower  temperatures  in  cold-blooded  as  com- 
pared to  warm-blooded  animals  is  due,  according  to  v.  Furth,  to  the  fact 
that  in  the  first  a  soluble  myogen  fibrin  occurs  preformed  in  the  muscle 
which  coagulates  at  30-40°  C,  while  in  the  warm-blooded  animals  the  coagu- 
lating substance  is  musculin  (myosin  of  v.  Furth)  which  coagulates  at  a 
higher  temperature.  Distilled  water  may  also  produce  a  rigor  in  the  muscles 
(water-rigor).  Acids,  even  very  weak  ones,  such  as  carbon  dioxide,  may 
quickly  produce  a  rigor  (acid-rigor),  or  hasten  its  appearance.  A  number 
of  chemically  different  substances,  such  as  chloroform,  ether,  alcohol, 
ethereal  oils,  caffeine,  and  many  alkaloids,  produce  a  similar  effect.  The 
rigor  which  is  produced  by  means  of  acids  or  other  agents  which,  like  alcohol, 
coagulate  proteids  must  be  considered  as  produced  by  entirely  different 
processes  from  those  causing  spontaneous  rigor. 

When  the  muscle  passes  into  rigor  mortis  it  becomes  shorter  and  thicker, 
harder  and  non-transparent,  and  less  ductile.  The  acid  part  of  the  ampho- 
teric reaction  becomes  stronger,  which  is  explained  by  most  investigators  by 
a  formation  of  lactic  acid.  There  is  hardly  any  doubt  that  this  increase  in 
acidity  may  at  least  in  part  be  due  to  a  transformation  of  a  part  of  the 
diphosphate  into  monophosphate  by  the  lactic  acid.  The  statements  in 
regard  to  the  presence  or  absence  of  free  lactic  acid  in  the  rigor-mortis 
muscle  are  contradictory.1     Besides  the  formation  of  acid,  the  chemical 

1  It  is  impossible  to  enter  into  the  details  of  the  disputed  statements  as  to  the  reaction 
of  the  muscles,  etc.  We  shall  only  refer  to  the  works  of  Rohmann,  Pfluger's  Arch., 
50  and  55,  and  Heffter,  Arch.  f.  exp.  Path.  u.  Pharm.,  31  and  38.  These  works  con- 
tain also  the  researches  of  the  older  investigators  more  or  less  completely. 


394  MUSCLE. 

processes  which  take  place  in  rigor  of  the  muscles  are  the  following:  By 
the  coagulation  of  the  plasma  a  myosin-clot  is  produced  which  is  the  cause 
of  the  hardening  and  of  the  diminished  transparency  of  the  muscle,  but  this 
view  must  be  changed  on  account  of  the  researches  of  v.  Furth,  which  have 
shown  that  the  clot  consists  of  myogen-  and  myosin-fibrin.  The  appear- 
ance of  this  clot  may  be  hastened  by  the  simultaneous  occurrence  of  lactic 
acid.  Carbon  dioxide  is  also  formed,  which  does  not  seem  to  be  a  direct 
oxidation  product,  but  a  product  of  the  cleavage  processes.  Hermann  l 
claims  that  carbon  dioxide  is  produced  in  the  removed  muscle,  even  in  the 
absence  of  oxygen,  when  it  passes  into  rigor  mortis.  In  connection  with 
this  view  we  must  call  attention  to  Folin  's  2  observations  that  no  proteid 
coagulation  took  place  in  rigor  under  special  conditions. 

As  many  investigators  admit  of  an  increased  formation  of  lactic  acid  on 
the  appearance  of  rigor  mortis,  the  question  arises,  from  what  constituents 
of  the  muscle  is  this  acid  derived?  The  most  probable  explanation  is  that 
the  lactic  acid  is  produced  from  the  glycogen,  as  certain  investigators,  such 
as  Nasse  and  Werther,  have  observed  a  decrease  in  the  quantity  of 
glycogen  in  rigor  of  the  muscle.  On  the  other  side,  Bohm  3  has  observed 
cases  in  which  no  consumption  of  glycogen  took  place  in  rigor  of  the  muscle, 
and  he  has  also  found  that  the  quantity  of  lactic  acid  produced  is  not  pro- 
portional to  the  quantity  of  glycogen.  It  is  therefore  possible  that  the 
consumption  of  glycogen  and  the  formation  of  lactic  acid  in  the  muscles 
are  two  processes  independent  of  each  other,  and,  as  above  stated  in  regard 
to  the  formation  of  paralactic  acid,  the  lactic  acid  of  the  muscle  may  be 
considered  as  a  decomposition  product  of  proteid.  The  origin  of  the  carbon 
dioxide  is  also  not  to  be  sought  for  in  the  decomposition  of  the  glycogen  or 
dextrose.  Pfluger  and  Stintzing  4  have  found  that  in  the  muscle  a  sub- 
stance occurs  which  evolves  large  quantities  of  carbon  dioxide  on  boiling 
with  water,  and  it  is  probably  this  substance  which  is  decomposed  with  the 
formation  of  carbon  dioxide  in  tetanus  as  well  as  in  rigor.  .  In  this  connec- 
tion we  call  attention  to  the  fact  that  phosphocarnic  acid  yields  lactic  acid 
as  well  as  carbon  dioxide  as  cleavage  products. 

After  the  muscles  have  been  rigid  for  some  time  they  relax  again  and 
the  muscles  become  softer.  This  is  in  part  produced  by  the  strong  acid 
dissolving  the  myosin-clot  and  in  part  to  autolytic  processes  (Vogel  5). 

Metabolism  in  the  Inactive  and  Active  Muscles.  It  is  admitted  by  a 
number  of  prominent  investigators,  Pfluger  and  Colasanti,  Zuntz  and 

1  "  Untersuchungen  iiber  den  Stoffwechsel  der  Muskeln,"  etc.     Berlin,  1867 

2  Amer.  Journ.  of  Physiol.,  9. 

3  Nasse,  Beitr.  z.  Physiol,  der  kontrakt.  Substanz,  Pfluger's  Arch.,  2;  Werther, 
ibid.,  40;   Bohm,  ibid.,  23  and  4G. 

*  Pfluger's  Arch.,  18. 

5  R.  Vogel,  Unters.  iiber  Muskelsaft,  Deutsch.  Arch.  f.  klin.  Med.,  1902. 


METABOLISM  IN   THE  MUSCLES. 

Rohrig,1  and  others,  that  the  metabolism  in  (he  muscles  is  regulated  by 
the  nervous  system.  When  at  rest,  when  there  is  qo  mechanical  exertion, 
there  exists  a  condition  which  Zi  my.  and  Rohrig  have  designated  "cfu  mical 

tonus."  This- tonus  seems  to  be  a  reflex  tonus,  for  it  may  be  reduced 
by  discontinuing  the  connection  between  the  muscles  and  the  central 
organ  of  the  nervous  system  by  cutting  through  the  spinal  cord  or  the 
muscle-nerves.  The  possibility  of  reducing  the  chemical  tonus  of  the 
muscles  in  various  ways  offers  an  important  means  of  deciding  the 
extent  and  kind  of  chemical  processes  going  on  in  the  muscles  when  at  rest. 
In  comparative  chemical  investigation  of  the  processes  in  the  active  and  the 
inactive  muscles  several  methods  of  procedure  have  been  adopted.  The 
same  active  and  inactive  muscles  have  been  compared  after  removal,  also 
the  arterial  and  venous  muscle-blood  in  rest  and  activity,  and  lastly  the 
total  exchange  of  material,  the  receipts  and  expenditures  of  the  organism, 
have  been  investigated  under  these  two  conditions. 

By  investigations  according  to  these  several  methods  it  has  been  found 
that  the  active  muscle  takes  up  oxygen  from  the  blood  and  returns  to  it 
carbon  dioxide,  and  also  that  the  quantity  of  oxygen  taken  up  is  greater 
than  the  oxygen  contained  in  the  carbon  dioxide  eliminated  at  the  same 
time.  The  muscle,  therefore,  holds  in  some  form  of  combination  a  part  of 
the  oxygen  taken  up  while  at  rest.  During  activity  the  exchange  of  mate- 
rial in  the  muscle,  and  therewith  the  exchange  of  gas,  is  increased.  The 
animal  organism  takes  up  much  more  oxygen  in  activity  than  when 
at  rest,  and  eliminates  also  considerably  more  carbon  dioxide.  The  quan- 
tity of  oxygen  which  leaves  the  body  as  carbon  dioxide  during  activity 
is  much  larger  than  the  quantity  of  oxygen  taken  up  at  the  same  time; 
and  the  venous  muscle-blood  is  poorer  in  oxygen  and  richer  in  carbon 
dioxide  during  activity  than  during  rest.  The  exchange  of  gases  in  the 
muscles  during  activity  is  the  reverse  of  that  at  rest,  for  the  active  muscle 
gives  up  a  quantity  of  carbon  dioxide  which  does  not  correspond  to  the 
quantity  of  oxygen  taken  up,  but  is  considerably  greater.  It  follows  from 
this  that  in  muscular  activity  not  only  does  oxidation  take  place,  but  also 
splitting  processes  occur.  This  results  also  from  the  fact  that  removed 
blood-free  muscles  when  placed  in  an  atmosphere  devoid  of  oxygen  can 
labor  for  some  time  and  also  yield  carbon  dioxide  (Hermann  2). 

During  muscular  inactivity,  in  the  ordinary  sense,  a  consumption  of 
glycogen  takes  place.  This  is  inferred  from  the  observations  of  several 
investigators  that  the  quantity  of  glycogen  is  increased  and  its  correspond'- 

1  Sep  the  works  of  Pfliiger  and  his  pupils  in  Pfliiger's  Arch.,  4.  12,  1 1,  16,  and  18- 
Rohrig,  ibid.,  4.  See  also  Zuntz,  ibid.,  12.  In  regard  to  the  metabolism  alter  curare 
poisoning,  see  also  Frank  and  Voit,  Zeitschr.  f.  Biologie,  42,  and  Frank  and  Geb- 
hard,  ibid.,  43. 

1  L.  c.  In  regard  to  gas  exchange  in  removed  muscles,  see  also  J.  Tissot,  Arch,  de 
Physiol.  (5),  6  and  7,  and  Compt.  rend.,  120. 


396  MUSCLE. 

ino-  consumption  reduced  in  those  muscles  whose  chemical  tonus  is 
reduced  either  by  cutting  through  the  nerve  or  for  other  reasons  (Bernard, 
Chandelon,  Way,1  and  others).  In  activity  this  consumption  of  glycogen 
is  increased,  and  it  has  been  positively  proved  by  the  researches  of  several 
investigators  (Nasse,  Weiss,  Kulz,  Marcuse,  Manche,  Morat  and  Dtjfotjr  2) 
that  the  quantity  of  glycogen  in  the  muscles  in  activity  decreases  quickly 
and  freely.  As  shown  by  the  researches  of  Chauveau  and  Kaufmann, 
Quixquaud,  Morat  and  Dufour,  Cavazzani,  and  especially  those  of 
Seegen,3  the  sugar  is  removed  from  the  blood  and  consumed  during  ac- 
tivity. According  to  Seegen  a  very  abundant  formation  of  sugar  takes 
place  in  the  liver,  and  correspondingly  the  blood  of  the  hepatic  vein  is 
much  richer  in  sugar  than  that  in  the  portal  vein;  and  this  sugar  of  the 
blood  is,  according  to  him,  the  source  of  heat  formation  and  mechanical 
activity.  It  is  nevertheless  true  that  important  objections  have  been 
presented  against  a  few  of  these  investigations,  and  a  sugar  formation, 
according  to  Seegen 's  idea,  has  been  denied  by  several  investigators, 
and  recently  by  Zuntz  and  Mosse;  4  but  still  there  can  exist  hardly  any 
doubt  that  sugar  is  consumed  in  muscular  activity. 

The  amphoteric  reaction  of  the  inactive  muscles  is  changed  during 
activity  to  an  acid  reaction  (Du  Bois-Reymond  and  others),  and  the  acid 
reaction  increases  to  a  certain  point  with  the  work.  The  quickly  contract- 
ing pale  muscles  produce,  according  to  Gleiss,5  more  acid  during  activity 
than  the  more  slowly  contracting  red  muscles.  The  acid  reaction  appearing 
durino-  activity  was  formerly  considered  to  be  due  to  the  formation  of  lactic 
acid,  a  view  which  has  been  contradicted  by  Astaschewsky,  Pfluger  and 
Warren,6  who  found  less  lactic  acid  in  the  tetanized  muscle  than  when  at 
rest.  Monari  also  found  a  decrease  in  the  quantity  of  lactic  acid  during 
activity,  and  according  to  Heffter  the  quantity  of  lactic  acid  in  the  muscle 
is  diminished  in  tetanus  produced  by  poison.  Contrary  to  these  investiga- 
tions Marcuse  and  Werther  7  have  been  able  to  prove  the  formation  of 


Chandelon,  Pfluger 's  Arch.,  13;  Way,  Arch.  f.  exp.  Path.  u.  Pharm.,  34,  which 
also  contains  the  pertinent  literature. 

2  Nasse,  Pfluger's  Arch.,  2;  Weiss,  Wien.  Sitzungsber. ,  64;  Kulz,  in  Ludwig's 
Festschrift,  Marburg,  1891;  Marcuse,  Pfluger's  Arch.,  39;  Manche\  Zeitschr.  f.  Biolo- 
gie,  25;   Morat  and  Dufour,  Arch,  de  Physiol.  (5),  4. 

3  Chauveau  and  Kaufmann,  Compt.  rend.,  103,  104,  and  105;  Quinquaud,  Maly's 
Jahresber.,  10;  Morat  and  Dufour,  1.  c;  Cavazzani,  Centralbl.  f.  Physiol.,  8;  Seegen, 
"Die  Zuckerbildung  im  Thierkorper, "  Berlin,  1890,  Centralbl.  f  Physiol.,  8,  9,  and 
10;   Du  Bois-Reymond 's  Arch.,  1895  and  1896;  Pfluger's  Arch.,  50. 

4  Mosse,  Pfluger's  Arch.,  03;  Zuntz,  Centralbl.  f.  Physiol.,  10,  and  Du  Bois- 
Reymond 's  Arch.,  1896,  538.     See  also  Schenck,  Pfluger's  Arch.,  61  and  65. 

5  Pfluger's  Arch.,  41. 

8  Astaschewsky,  Zeitschr.  f.  physiol.  Chem.,  4;  Warren,  Pfluger's  Arch.,  24. 
7  Monari,   Maly's  Jahresber.,    19;    Heffter,   Arch.    f.    exp.    Path.  u.    Pharm.,  31j 
Marcuse,  1.  c;    Werther,  Pfluger's  Arch.,  46. 


METABOLISM  IN   THE  MUSCLES.  397 

lactic  acid  during  activity;    still  the  statements  are  very  contradictory 
Other  observations  speak  for  a  formation  of  lactic  acid  during  activity. 

Thus  SPIRO  found  an  increase  in  the  quantity  of  lactic  acid  in  the  blood 
during  work.  Colasanti  and  Moscatelli  found.small  quantities  of  lactic 
acid  in  human  urine  after  strenuous  marches,  and  Werther  observed  an 
abundance  of  lactic  acid  in  the  urine  of  frogs  after  tetanization.  According 
to  Hoppe-Seylbb,  on  the  contrary,  in  agreement  with  his  view  in  regard 
to  the  formation  of  lactic  acid,  lactic  acid  is  not  produced  regularly  during 
work,  but  only  when  insufficient  oxygen  is  supplied.  Zillesen  l  has  also 
found  that  on  artificially  cutting  off  the  oxygen  from  the  muscles  during 
life  more  lactic  acid  was  formed  than  under  normal  conditions. 

It  is  evident  that  the  experiments  with  the  muscles  in  situ — in  other 
words,  with  muscles  through  which  blood  is  passing — cannot  yield  any  con- 
clusion to  the  above  question,  as  the  lactic  acid  formed  during  wrork  may 
perhaps  be  removed  by  the  blood.  The  following  objections  can  be  made 
against  those  experiments  in  which  lactic  acid  has  been  found  after  mod- 
erate work  in  the  blood  or  the  urine,  as  also  especially  against  the  experi- 
ments with  removed  active  muscles,  namely,  that  in  these  cases  the  supply 
of  oxygen  to  the  muscles  was  not  sufficient,  and  that  the  lactic  acid  formed 
thereby  is  not,  in  accordance  with  the  views  of  Hoppe-Seyler,  a  perfectly 
normal  process.  The  question  as  to  the  formation  of  lactic  acid  in  the 
active  muscle  under  perfect  physiological  conditions  is  still  an  open  one, 
although  several  observations  make  it  seem  as  if  it  wras  very  probable. 

According  to  Siegfried  the  amount  of  phosphocarnic  acid  is  dimin- 
ished during  activity.  Macleod  claims  that  this  is  only  true  for  intense 
muscular  activity,  while  otherwise  with  work  the  organic  phosphorus 
not  present  as  nucleons  is  diminished  and  the  quantity  of  phosphates 
is  increased.  It  stands  in  accord  with  Weyl  and  Zeitler's  2  observa- 
tions that  the  active  muscle  contains  more  phosphoric  acid  than  the  inac- 
tive muscle.  As  in  the  dead  muscle,  so  in  the  active  muscle,  the  some- 
what stronger  acid  reaction  is  in  part  due  to  a  greater  quantity  of  mono- 
phosphate. 

The  amount  of  proteids  in  the  removed  muscles  is,  according  to  the 
older  investigators,  decreased  by  work.  The  correctness  of  this  statement 
is,  however,  disputed  by  other  investigators.  The  older  statements  in 
regard  to  the  nitrogenous  extractive  bodies  of  the  muscle  in  rest  and  in 
activity  are  likewise  uncertain.  According  to  the  recent  researches  of 
Monari  3  the  total  quantity  of  creatine  and  creatinine  is  increased  by 


!Spiro,  Zeitschr.  f.  physiol.  Chem.,  1;  Colasanti  and  Moscatelli,  Italy's  Jahresber., 
17,  212;  Hoppe-Seyler,  1.  c,  and  Zeitschr.  f.  physiol.  Chem.,  19;   Zillesen,  ibid.,  16 

1  Siegfried,  Zeitschr.  f.  physiol.  Chem.,  21;  Macleod,  ibid.,  2S;  Weyl  and  Zeitler, 
ibid.,  16. 

'Maly's  Jahresber.,  19,  296. 


39S  MUSCLE. 

work,  and  indeed  the  amount  of  creatinine  is  especially  augmented  by  an 
excess  of  muscular  activity.  The  creatinine  is  formed  essentially  from 
the  creatine.  In  excessive  activity  Monari  also  found  xantho-creatinine 
in  the  muscle,  and  the  quantity  was  one-tenth  that  of  the  creatinine. 
The  quantity  of  xanthine  bodies  is,  according  to  Monari,  decreased  under 
the  influence  of  work.  It  seems  to  have  been  positively  shown  that  the 
active  muscle  contains  a  smaller  quantity  of  bodies  soluble  in  water  and 
a  larger  quantity  of  bodies  soluble  in  alcohol  than  the  resting-muscle 
(Helmholtz  *). 

Attempts  have  been  made  to  solve  the  question  relative  to  the  behavior 
of  the  nitrogenized  constituents  of  the  muscle  at  rest  and  during  activity  by 
determining  the  total  quantity  of  nitrogen  eliminated  under  these  different 
conditions  of  the  body.  While  formerly  it  was  held  with  Liebig  that  the 
elimination  of  nitrogen  by  the  urine  was  increased  by  muscular  work,  the 
researches  of  several  experimenters,  especially  those  of  Voit  on  dogs  and 
Pettexkofer  and  Voit  on  men,  have  led  to  quite  different  results.  They 
have  shown,  as  has  also  lately  been  confirmed  by  other  investigators, 
especially  I.  Munk  and  Hirschfeld,2  that  during  work  no  increase  or 
only  a  very  insignificant  increase  in  the  elimination  of  nitrogen  takes  place. 

We  should  not  omit  to  mention  the  fact  that  a  series  of  experiments 
has  been  made  showing  a  significant  increase  in  the  metabolism  of  proteids 
during  or  after  work.  There  are  for  example  the  observations  of  Flint  and 
of  Pavy  on  a  pedestrian,  v.  Wolff,  v.  Funke,  Kreuzhage  and  Kellxer  on 
a  horse,  and  Dunlop  and  his  collaborators  on  working  human  beings,  and 
of  Krummacher,  Pfluger,  Zuntz  and  his  pupils,3  and  others.  The  re- 
searches on  the  elimination  of  sulphur  during  rest  and  activity  also  belong 
to  this  category.  The  elimination  of  nitrogen  and  sulphur  runs  parallel 
with  the  metabolism  of  proteids  in  resting  and  active  persons,  and  the  quan- 
tity of  sulphur  excreted  by  the  urine  is  therefore  also  a  measure  of  the  pro- 
teid  decomposition.  The  older  researches  of  Engelmann,  Flint,  and  Pavy, 
as  well  as  the  more  recent  ones  of  Beck  and  Benedict,4  and  Dunlop  and 
his  collaborators,  show  an  increased  elimination  of  sulphur  during  or  after 
work,  and  this  speaks  for  an  increased  proteid  metabolism  because  of  mus- 
cular activity. 

1Arch.  f.  Anat.  u.  Physiol.,  1845. 

2  Voit,  Untersuchungen  iiber  den  Einfluss  des  Kochsalzes,  des  Kaffees  und  der 
Muskelbewegungen  auf  den  Stoffwechsel  (Miinchen,  I860),  and  Zeitschr.  f.  Biologie,  2; 
I.  Munk,  Du  Bois-Reymond's  Arch.,  1890  and  1896;  Hirschfeld,  Virchow's  Arch  ,  121. 

3  Flint,  Journ.  of  Anat.  and  Physiol.,  11  and  12;  Pavy,  The  Lancet,  1876  and  1877; 
v.  Wolff,  v.  Funke,  Kellner,  cited  from  Voit,  Hermann's  Handb.,  C,  197;  Dunlop,  Noel- 
Paton,  Stockman,  and  Maccadam,  Journ.  of  Physiol.,  22;  Kummacher,  Zeitschr.  f. 
Biologie,  33;   Pfluger,  Pfliiger's  Arch.,  50;    Zuntz,  Arch.  f.  (Anat.  u.)  Physiol.,  1894. 

4  Engelmann,  ibid.,  1871;  Beck  and  Benedict,  Pfliiger's  Arch.,  54,  and  also  foot- 
note 2. 


METABOLISM  AND  MUSCULAR  activity.  399 

'That  an  increased  destruction  of  proteid  is  not  necessarily  produced  by 
work  follows  from  the  recent  observations  of  Caspaei,  Bornstein,  K 
Waits,  A.  Lobwt,  A.twateb  and  Benedict,1  that  a  relent  ion  of  nitrogen 
and  a  deposition  of  proteid  occurs  during  work.  The  contradictory  obser- 
vations on  the  proteid  destruction  during  and  caused  by  work  are  not 
directly  in  opposition  to  each  other,  because  the  extent  of  proteid  metabo- 
lism is  dependent  upon  many  conditions,  such  as  the  quantity  and  composite  « 
of  the  food,  the  condition  of  the  adipose  tissue  of  the  body,  the  action  of 
the  work  upon  the  respiratory  mechanism,  etc.,  all  of  which  have  an 
influence  on  the  results  of  the  experiments. 

Recently  Steyrer  2  has  found  that  the  muscle  juice  of  a  continuously  totanized 
muscle  was  somewhat  poorer  in  musculin  and  correspondingly  richer  in  myogen 
than  the  juice  from  a  similar  non-tetanized  muscle.  We  cannot  draw  any  con- 
clusions from  this  experiment,  but  it  seems  to  show  that  the  proteids  are  not 
consumed  in  work. 

The  older  investigations  on  the  amount  of  fat  in  muscles  removed  after 
activity  and  after  rest  have  not  led  to  any  definite  results.  According  to  the 
recent  investigations  of  Zuntz  and  Bogdanow  3  the  fat  belonging  to  the 
muscle-fibres  and  which  is  difficultly  extracted  takes  part  in  work.  Besides 
these  there  are  several  researches  by  Voit,  Pettenkofer  and  Voit,  J. 
Frentzel,4  and  others  which  make  an  increased  destruction  of  fat  during 
work  probable. 

If  the  results  of  the  investigations  thus  far  made  of  the  chemical  proc- 
esses going  on  in  the  active  and  inactive  muscle  were  collected  together,  we 
would  find  the  following  characteristics  for  the  active  muscle.  The  active 
muscle  takes  up  more  oxygen  and  gives  off  more  carbon  dioxide  than  the 
inactive  muscle;  still  the  elimination  of  carbon  dioxide  is  increased  con- 
siderably more  than  the  absorption  of  oxygen.     The  respiratory  quotient, 

CO 

— p,  is  found  to  be  regularly  raised  during  work;  yet  this  rise,  which  will 

be  explained  in  detail  in  a  following  chapter  on  metabolism,  can  hardly  be 
conditioned  on  the  kind  of  processes  going  on  in  the  muscle  during  activity 
with  a  sufficient  supply  of  oxygen.  In  work  a  consumption  of  carbohydrates, 
glycogen,  and  sugar  takes  place.  A  consumption  of  sugar  seems  only  to 
have  been  shown  in  muscle  with  blood  circulation,  while  a  consumption  of 
glycogen  also  has  been  observed  in  removed  muscle.  The  acid  reaction  of 
the  muscle  becomes  greater  with  work.     In  regard  to  the  extent  of  a  re- 


1  Caspari,  Pfliiger's  Arch.,  83;  Bornstein,  ibid.',  Kaup,  Zeitschr.  f.  Biologie,  43; 
Waite,  U.  S.  Depart.  Agricult.  Bulletin  S9  (1901);  Atwater  and  Benedict,  ibid., 
Bull.  G9  (1899);  Loewy,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 

1  Hof meister  's  Beitriige,  4. 

s  Arch.  f.  (Anat.  u.)  Physiol.,  1897. 

4  Pfliiger's  Arch.,  68. 


400  MUSCLE. 

formation  of  lactic  acid  opinion  is  divided.  An  increased  consumption 
of  fat  has  occasionally  been  observed.  The  quantity  of  organic  phos- 
phorus decreases,  and  an  increase  in  the  nitrogenous  extractives  of  the 
creatinine  group  seems  also  to  occur.  Proteid  metabolism  has  been  found 
increased  in  certain  series  of  experiments,  and  not  in  others;  but  an  in- 
creased elimination  of  nitrogen  as  a  direct  consequence  of  muscular  exertion 
has  thus  far  not  been  positively  proved. 

In  close  connection  -with  the  above-mentioned  facts  there  is  the  question 
as  to  the  origin  of  muscular  activity  so  far  as  it  has  its  origin  in  chemical 
processes.  In  the  past  the  generally  accepted  opinion  was  that  of  Liebig. 
that  the  source  of  muscular  action  consisted  of  a  metabolism  of  the  proteid 
bodies:  to-day  another,  generally  accepted,  view  prevails.  Fick  and  Wis- 
iiCExrs  '  climbed  the  Faulhorn  and  calculated  the  amount  of  mechanical 
force  expended  in  the  attempt.  With  this  they  compared  the  mechanical 
equivalent  transformed  in  the  same  time  from  the  proteids,  calculated  from 
the  nitrogen  eliminated  with  the  urine,  and  found  that  the  work  really 
performed  was  not  by  any  means  compensated  by  the  consumption  of 
proteid.  It  was  therefore  proved  by  this  that  proteids  alone  cannot  be  the 
source  of  muscular  activity,  and  that  this  depends  in  great  measure  on  the 
metabolism  of  non-nitrogenous  substances.  Many  other  observations  have 
led  to  the  same  result,  especially  the  experiments  of  Voit,  of  Pettexkofer 
and  Voit,  and  of  other  investigators,  whose  observations  show  that  while 
the  elimination  of  nitrogen  remains  unchanged,  the  elimination  of  carbon 
dioxide  during  work  is  very  considerably  increased.  It  is  also  generally 
considered  as  positively  proved  that  muscular  work  is  produced,  at  least  in 
greatest  part,  by  the  metabolism  of  non-nitrogenous  substances.  Never- 
theless there  is  no  warrant  for  the  statement  that  muscular  activity  is  pro- 
duced entirely  at  the  cost  of  the  non-nitrogenous  substances,  and  that  the 
proteid  bodies  are  without  importance  as  a  source  of  energy. 

The  investigations  of  Pfluger  2  are  of  great  interest  in  this  connection. 
He  fed  a  bulldog  for  more  than  seven  months  with  meat  which  alone  did  not 
contain  sufficient  fat  and  carbohydrates  even  for  the  production  of  heart 
activity,  and  then  let  him  work  very  hard  for  periods  of  14,  35,  and  41 
days.  The  positive  results  obtained  by  these  series  of  experiments  was  that 
"complete  muscular  activity  may  be  effected  to  the  greatest  extent  in  the 
absence  of  fat  and  carbohydrates, ' '  and  the  ability  of  proteids  to  serve  as  a 
source  of  muscular  energy  cannot  be  denied. 

The  nitrogenous  as  well  as  the  non-nitrogenous  nutriments  may  serve  as 
a  source  of  energy ;  but  the  views  are  divided  in  regard  to  the  relative  value 
of  these.     Pfluger  claims  that  no  muscular  work  takes  place  without  a 

1  Vierteljahrsschr.  d.  Zurich,  riaturf.  Gesellsch.,  10.     Cited  from  Centralbl.  f.  d. 
med.  WIss.,  1866,  309. 
J  Pfluger 's  Arch,  50. 


QUANTITATIVE  COMPOSITION  OF   THE  MUSCLE.  401 

decomposition  of  proteid,  and  the  living  cell-substance  prefers  always  the 
proteid  and  rejects  the  fat  and  sugar,  contenting  itself  with  these  only  when 
proteids  are  absent.  Other  investigators,  on  the  contrary,  believe  that  the 
muscles  first  draw  on  the  supply  of  non-nitrogenous  nutriments,  and  accord- 
ing to  Seegex,  Chauveau,  and  Laulaxie  '  the  sugar  is  indeed  the  only 
direct  source  of  muscular  force.  The  last-mentioned  investigator  holds 
that  the  fat  is  not  directly  utilized  for  work,  but  only  after  a  previous 
conversion  into  sugar.  Zuxtz  and  his  collaborators  have  made  strong 
objections  to  the  correctness  of  such  a  view.  If,  according  to  Zuxtz,  the 
fat  must  be  first  transformed  into  sugar  before  it  can  serve  as  source  of 
muscular  work,  it  must  require  about  30  per  cent  more  energy  to  perform 
the  same  work  with  fatty  food  as  it  does  with  carbohydrates;  but  this  is 
not  the  case.  The  investigations  of  Zuxtz  (together  with),  Loeb,  Heixe- 
manx,  Frextzel  and  Reach  2  show  that  all  foodstuffs  have  nearly  the  same 
power  of  serving  as  material  for  the  work  of  the  muscles.  The  law  of  the 
substitution  of  the  foodstuffs,  according  to  their  combustion  equivalents,  is 
also  true  for  muscular  work  and  fat  correspondingly  acts  with  its  full  amount 
of  energy  without  previously  being  transformed  into  sugar.  The  question 
which  foodstuff  the  muscle  prefers  is  dependent  upon  the  quantity  of  the 
same  at  the  disposal  of  the  muscle.  A  direct  substitution  of  the  body 
material  by  the  bodies  supplied  as  food  does  not  take  place  in  the  muscular 
activity  in  the  ordinary  nutritive  condition.  According  to  Johansson  and 
Koraex  3  the  C02  excretion  produced  by  certain  work  is  not  influenced  by 
the  supply  of  foodstuffs  (proteid  or  sugar). 

Siegfried  considers,  as  above  stated,  the  phosphocarnic  acid  as  a  source  of 
energy.  According  to  his  and  KrI'ger 's  *  researches  phosphocarnic  acid,  which 
yields  on  cleavage,  among  other  bodies,  carbon  dioxide,  occurs  in  part  preformed 
in  the  muscle,  and  in  part  as  a  hypothetical  aldehyde  combination  of  the  same — a 
combination  which  forms  phosphocarnic  acid  on  oxidation.  Siegfried  therefore 
makes  the  suggestion  that  in  the  resting  muscle,  which  requires  more  oxygen 
than  exists  in  the  carbon  dioxide  eliminated,  this  reducing  aldehyde  substance  is 
gradually  oxidized  to  phosphocarnic  acid,  which  is  used  in  the  activity  of  the 
muscle  with  the  split  tin    off  of  carbon  dioxide. 

Quantitative  Composition  of  the  Muscle.  A  large  number  of  analvses 
have  been  made  of  the  flesh  of  various  animals  for  puiely  practical  purposes, 
in  order  to  determine  the  nutritive  value  of  different  varieties  of  meat ;  but 
there  are  no  exact  scientific  analyses  with  sufficient  regard  to  the  quantity  of 

1  See  Seegen,  foot-note  3,  page  396.  The  works  of  Chauveau  and  his  collaborators 
are  found  in  Compt.  rend.,  121,  122,  and  123;  Laulanie,  Arch,  de  PhysioL  (5),  S. 

'  Loeb,  Arch.  f.  (Anat.  u.)  Physiol.,  1894j  Heinemann,  Pfliiger's  Arch.,  S3;  Frentzel 
and  Reach,  ibid. 

?>kand.  Arch.  f.  Physiol.,  13. 

4  Zeitschr.  f.  physiol.  Chem.,  22. 


Muscles  of 
Birds. 

Muscles  of 

Cold-blooded 

Animals. 

227-282 

200 

717-773 

800 

217-263 

180-190 

10-19 

10-20 

29.8-111 

29.7-87 

88.0-184 

70.0-121 

3.4 

2.3 

0.7-1.3 

— 

0.1-0.3 

— 

— 

7.0 

— 

1.1 

402  MUSCLE. 

different  proteid   bodies  and  the  remaining  muscle-constituents,  or  these 
analyses  are  incomplete  or  of  little  value. 

To  give  the  reader  some  idea  of  the  variable  composition  of  muscle- 
substance  the  following  summary  is  presented,  chiefly  obtained  from  K.  B. 
Hofmann  \s  i  book.    The  figures  are  parts  per  1000. 

Muscles  of 

Mammals. 

Solids 217-255 

Water 74.5-783 

Organic  bodies 208-245 

Inorganic  bodies 9-10 

Myosin 35-106 

Stroma  substance  (Danilewsky) 78-161 

Creatine 2 

Xanthine  bodies 1 . 3-1 . 7 

Inosinic  acid  (barium  salt) 0.1 

Protic  acid — 

Taurin 0.7  (horse) 

Inosite 0.03 

Glycogen 4-37                          —                           3-5 

Lactic  acid 0 . 4-0 .7                       —                            — 

Phosphoric  acid 3 . 4-4 . 8 

Potash 3.0-4.0 

Soda 0.3 

Lime 0.2 

Magnesia 0.4 

Sodium  chloride 0 .  04-0 . 1 

Iron  oxide 0.04-0.1 

In  this  table,  which  has  little  value  because  of  the  variation  in  the  com- 
position of  the  muscles,  no  results  are  given  as  to  the  estimates  of  fat.  Owing 
to  the  variable  quantity  of  fat  in  meat  and  the  incompleteness  of  the  older 
methods  of  estimation  it  is  hardly  possible  to  quote  a  positive  average  for 
this  body.  After  most  careful  efforts  to  remove  the  fat  from  the  muscles 
without  chemical  means,  it  has  been  found  that  a  variable  quantity  of  inter- 
muscular fat,  which  does  not  really  belong  to  the  muscular  tissue,  always 
remains.  The  smallest  quantity  of  fat  in  the  muscles  from  lean  oxen  is 
6.1  p.  m.  according  to  Grouven,  and  7.6  p.  m.  according  to  Petersen. 
This  last  observer  also  found  regularly  a  smaller  quantity  of  fat,  7.6-8.6 
p.  m.,  in  the  fore  quarters  of  oxen,  and  a  greater  amount,  30.1-34.6  p.  m., 
in  the  hind  quarters  of  the  animal,  but  this  could  not  be  substantiated 
by  Steil.2  A  small  quantity  of  fat  has  also  been  found  in  the  muscles 
of  wild  animals.  B.  Konig  and  Farwick  found  10.7  p.  m.  fat  in  the  muscles 
of  the  extremities  of  the  hare,  and  14.3  p.  m.  in  the  muscles  of  the  partridge. 
The  muscles  of  pigs  and  fattened  animals  are,  when  all  the  adherent  fat 
is  removed,  very  rich  in  fat,  amounting  to  40-90  p.  m.    The  muscles  of 

1  Lehrbuch  d.  Zoochem.  (Wien,  1876),  104. 

2  See  Steil,  Pfliiger's  Arch.,  61. 


COMPOSITION  OF  MUSCLES.  403 

certain  fishes  also  contain  a  large  quantity  of  fat.  According  to  Alme.v, 
in  the  flesh  of  the  salmon,  the  mackerel,  and  the  eel  there  are  contained 
respectively  100,  104,  and  329  p.  m.  fat.1 

The  quantity  of  water  in  the  muscle  is  liable  to  considerable  variation. 
The  quantity  of  fat  has  a  special  influence  on  the  quantity  of  water,  and  one 
finds,  as  a  rule,  that  the  flesh  which  is  deficient  in  water  is  correspondingly 
rich  in  fat.  The  quantity  of  water  does  not  depend  alone  upon  the  amount 
of  fat,  but  upon  many  other  circumstances,  among  which  musi  be  mentioned 
the  age  of  the  animal.  In  young  animals  the  organs  in  general,  and  there- 
fore also  the  muscles,  are  poorer  in  solids  and  richer  in  water.  In  man  the 
quantity  of  water  decreases  until  mature  age,  but  increases  again  towards 
old  age.  Work  and  rest  also  influence  the  quantity  of  water,  for  the  active 
muscle  contains  more  water  than  the  inactive.  The  uninterruptedly  active 
heart  should  therefore  be  the  muscle  richest  in  water.  That  the  quantity 
of  water  may  vary  independently  of  the  amount  of  fat  is  strikingly  shown 
by  comparing  the  muscles  of  different  species  of  animals.  In  cold-blooded 
animals  the  muscles  generally  have  a  greater  quantity  of  water,  in  birds  a 
lower.  The  comparison  of  the  flesh  of  cattle  and  fish  shows  very  strikingly 
the  different  amounts  of  water  (independent  of  the  quantity  of  fat)  in  the 
flesh  of  different  animals.  According  to  the  analysis  of  Almen,2  the  muscles 
of  lean  oxen  contain  15  p.  m.  fat  and  767  p.  m.  water;  the  flesh  of  the 
pike  contains  only  1.5  fat  and  839  p.  m.  water. 

For  certain  purposes,  as,  for  example,  in  experiments  on  metabolism,  it 
is  important  to  know  the  elementary  composition  of  flesh.  In  regard  to 
the  quantity  of  nitrogen  we  generally  accept  Voit's  figure,  namely,  3.4 
per  cent,  as  an  average  for  fresh  lean  meat.  According  to  Nowak  and 
Huppert  3  this  quantity  may  vary  about  0.6  per  cent,  and  in  more  exact 
investigations  it  is  therefore  necessary  to  specially  determine  the  nitrogen. 
Complete  elementary  analyses  of  flesh  have  recently  been  made  with  great 
care  by  Argutinsky.  The  average  for  ox-flesh  dried  in  vacuo  and  free 
from  fat  and  with  the  glycogen  deducted  was  as  follows:  C  49.6;  H  6.9; 
N  15.3;  O+S  23.0;  and  ash  5.2  per  cent.  Kohler  found  as  an  average  for 
water  and  fat-free  beef  C  49.86;  H  6.78;  N  15.68;  O+S  22.3  per  cent,  which 
are  very  similar  results.  This  investigator  has  also  made  similar  analyses 
of  the  flesh  of  various  animals  and  has  also  determined  the  calorific  value 
of  the  ash  and  fat-free  dried  meat  substance.  This  value  was,  per  gram 
of  substance,  5.599-5.677  cal.    The  relationship  of  the  carbon  to  nitrogen, 

1  In  regard  to  the  literature  and  complete  statements  on  the  composition  of  flesh 
of  various  animals,  see  Konig,  Chemie  der  menschlichen  Xahrungs-  und  Genussmittel, 
4.  Aufl. 

2  Nova  Act.  reg.  Soc.  Scient.  Upsal.,  Vol.  extr.  ord.,  1877;  also  Maly 's  Jahresber.,  7. 
sVoit,  Zeitschr.  f.  Biologic,  1;   Huppert,  ibid.,  7;  Nowak,  Wien.  Sitzungsber.,  W, 

Abth.  II. 


404  MUSCLE. 

which  Argutinsky  calls  the  "flesh  quotient,"  is  on  an  average  3.24  :1. 
From  Kohler's  analyses  the  average  for  beef  is  3.15  : 1  and  for  horse-flesh 
3.38  : 1.  According  to  Salkowski,  of  the  total  nitrogen  of  beef  77.4  per 
cent  was  insoluble  proteids,  10.08  per  cent  soluble  proteids,  and  12.52  per 
cent  other  soluble  bodies.  Frentzel  and  Schreuer  1  find  that  about 
7.74  per  cent  of  the  total  nitrogen  belongs  to  the  nitrogenous  extractives. 

There  exist  complete  investigations  by  Katz  2  as  to  the  quantity  of  min- 
eral constituents  of  the  muscles  from  man  and  animals.  The  variation  in 
the  different  elements  is  considerable.  Pork  is  much  richer  in  sodium  as 
compared  with  potassium  than  other  kinds  of  meat.  The  quantity  of  mag- 
nesium is  greater  and  often  considerably  greater  than  calcium  in  all  kinds  of 
flesh  investigated,  with  the  exception  of  shell-fish,  the  eel,  and  the  pike. 
Beef  is  very  poor  in  calcium.  Potassium  and  phosphoric  acid  are  the  most 
abundant  mineral  constituents  of  all  flesh. 

Non-striated  Muscles. 

The  smooth  muscles  have  a  neutral  or  alkaline  reaction  (Du  Bois- 
Reymond)  when  at  rest.  During  activity  they  are  acid,  which  is  inferred 
from  the  observations  of  Bernstein,  who  found  that  the  nearly  continually 
contracting  sphincter  muscle  of  the  Anodonta  is  acid  during  life.  The 
smooth  muscles  may  also,  according  to  Heidenhain  and  Kuhne,  pass  into 
rigor  mortis  and  thereby  become  acid.  A  spontaneous  but  slowly  coagulat- 
ing plasma  has  also  been  observed  in  several  cases. 

In  regard  to  the  proteids  of  the  smooth  muscles  we  have  the  older 
statements  of  Heidenhain  and  Hellwig;  3  they  were  first  carefully  studied 
according  to  newer  methods  by  Munk  and  Velichi.4  This  last  experi- 
menter has  prepared  a  neutral  plasma  from  the  gizzard  of  geese,  according 
to  v.  Furth's  method.  This  plasma  coagulated  spontaneously  at  the 
temperature  of  the  room,  although  slowly.  It  contained  a  globulin,  pre- 
cipitated by  dialysis,  which  coagulated  at  55-60°  C.  and  also  showed  cer- 
tain similarities  with  Kuhne 's  myosin.  A  spontaneously  coagulating 
albumin,  which  differed  from  myogen  (v.  Furth)  by  coagulating  at  45-50°  C, 
and  which  passes  by  spontaneous  coagulation  into  the  coagulated  modifica- 
tion without  a  soluble  intermediate  product,  exists  in  still  greater  quan- 
tities in  this  plasma.     Alkali  albuminates  do  not  occur,  but  a  nucleoproteid 

1  Argutinsky,  Pfliiger's  Arch.,  55;  Kohler,  Zeitschr.  f.  physiol.  Chem.,  31;  Sal- 
kowski, Centralbl.  f.  d.  med.  Wissensch.,  1894;  Frentzel  and  Schreuer,  Arch.  f.  (Anat. 
u.)  Physiol.,  1902. 

2  Pfliiger's  Arch.,  63.     See  also  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39. 

3  Du  Bois-Reymond  in  Xasse,  Hermann's  Handb.,  1,  339;  Bernstein,  ibid. ;  Heiden- 
hain, ibid,  340,  with  Hellwig,  ibid.,  339;   Kuhne,  Lehrbuch,  331. 

4  Munk  and  Velichi,  Centralbl.  f.  Physiol.,  12. 


NON-STRIATED  MUSCLES.  405 

is  found,  which  exists  in  about  five  times  the  quantity  as  compared  with 
striated  muscles. 

Recent  investigations  of  Bottazzi  and  Cappeli,  Vincent  and  Lewis, 
Vincent,  and  v.-Furth,1  some  on  the  muscles  of  warm-blooded  and  some 
on  those  of  lower  animals,  have  led  to  somewhat  contradictory  results,  but 
they  substantiate,  as  a  whole,  the  observations  of  Muxk  and  Velichi. 
Resides  the  nucleoproteids  the  smooth  muscles  contain  two  bodies  corre- 
sponding in  coagulation  temperature  to  musculin  and  myosinogen  (myogen, 
v.  Furth)  but  they  are  not  identical  therewith. 

llivmoglobin  occurs  in  the  smooth  muscles  of  certain  animals,  but  is 
absent  in  others.  Creatine  has  been  found  by  Lehmanx.2  According  to 
Fremy  and  Valenciennes8  the  muscles  of  the  cephalopods  contain 
taurin  besides  creatinine  {creatine?).  Of  the  non-nitrogenous  substances, 
glycogen  and  lactic  acid  have  been  found  without  doubt.  The  mineral  con- 
stituents show  th  •  remarkable  fact  that  the  sodium  combinations  exceed 
the  potassium  combinations. 

1  Bottazzi,  Centralbl.  f.  Physiol.,  15;  Vincent  and  Lewis,  Journ.  of  Physiol.,  26; 
Vincent,  Zeitschr.  f.  physiol.  Chem.,  34;  v.  Furth,  ibid.,  31. 

2  Cited  from  Nasse,  1.  c,  339. 

8  Cited  from  Kiihne's  Lehrbuch,  333. 


CHAPTER  XII. 
BRAIN  AND  NERVES. 

On  account  of  the  difficulty  in  making  a  mechanical  separation  and 
isolation  of  the  different  tissue-elements  of  the  nervous  central  organ  and 
the  nerves,  we  must  resort  to  a  few  microchemical  reactions,  chiefly  to 
qualitative  and  quantitative  investigations  of  the  different  parts  of  the 
brain,  in  order  to  study  the  varied  chemical  composition  of  the  cells  and 
the  nerve-axes.  This  study  is  accompanied  with  the  greatest  difficulty; 
and  although  our  knowledge  of  the  chemical  composition  of  the  brain  and 
nerves  has  been  somewhat  extended  by  the  investigations  of  modern  times, 
still  it  must  be  admitted  that  this  subject  is  as  yet  one  of  the  most  obscure 
and  complicated  in  physiological  chemistry. 

Proteids  of  different  kinds  have  been  shown  to  be  chemical  constituents 
of  the  brain  and  nerves.  Some  of  them  are  insoluble  in  water  and  dilute 
neutral-salt  solutions,  and  some  are  soluble  therein.  Among  the  latter  are 
found  albumin  and  globulin.  Nucleoalbumin,  which  is  often  considered  as 
an  alkali  albuminate,  also  occurs  in  the  brain.  Just  as  there  are  lecithin- 
albumins,  compounds  of  nucleoalbumins  with  lecithin,  so  according  to 
Ulpiani  and  Lelli  *  there  exists  an  analogous  compound  with  protagon  in 
the  brain  which  is  considered  by  these  experimenters  as  a  combination 
between  protagon  and  a  pseudonuclein.  Halliburton  2  found  two  globu- 
lins in  the  brain,  one  of  which  coagulated  at  47-50°  C.  and  the  other  at 
70°  C.  He  found  in  the  gray  matter  a  nucleoalbumin  which  coagulated 
at  55-60°  C.  and  contained  0.5  per  cent  phosphorus.  It  is  not  known 
what  relation  this  nucleoalbumin  bears  to  the  nucleoproteid  detected  by 
Levenb  a  which  contains  about  the  same  quantity  of  phosphorus,  namely 
0.56  per  cent.  This  last-mentioned  nucleoproteid  yields  adenine  and 
guanine  as  cleavage  products.  There  does  not  seem  to  be  any  doubt 
that  the  proteids  belong  chiefly  to  the  gray  substance  of  the  brain 
and    to    the   axis-cylinders.      The  same  remarks  apply  to   the  nuclein, 

'Cited  from  Chem.  Centralhl.,  1902,  2,  292. 

7  On  the  chemical  physiology  of  the  animal  cell,  Kings  College,  London,  Physio- 
logical Laboratory  Collected  Papers  No.  1,  1893. 

3  Arch,  of  Neurology  and  Psychopathology,  2  (1899). 

406 


CONSTITUENTS  OF   THE   HUMS  AND  NERVES.  407 

which  v.  Jacksch  '  found  in  large  quantities  in  the  gray  substance.  Neuro- 
keratin, which  was  first  detected  by  Ki  fauna,  and  which  partly  forms  the 
in  wroglia,  and  which  as  a  double  sheath  envelops  the  outside  of  the  nerve 
medulla  under  Schwann's  sheath  and  the  inner  axis-cylinders,  chiefly 
occurs  in  the  white  substance  (Kuhne  and  Chittenden,  Baumstark  2). 

The  phosphorized  substance  protagon  must  be  considered  as  one  of  the 
chief  constituents,  perhaps  the  only  constituent  (Baumstark),  of  the  white 
substance.  This  last-mentioned  substance,  if  we  keep  for  the  present  to 
the  must  carefully  studied  protagon — because  there  are  perhaps  several 
different  protagons — yields  as  decomposition  products  lecithin,  fatty  acids, 
and  a  nitrogenous  substance,  cerebrin.  It  is  difficult  to  state  whether 
this  hotly  also  exists  preformed  in  the  brain.  At  least  an  allied  substance, 
cerebron,  occurs  preformed  in  the  brain.  That  lecithin  also  is  pre-existent 
in  the  brain  and  nerves  can  hardly  be  doubted.  The  investigations  thus 
far  made  have  not  shown  decidedly  whether  it  is  more  abundant  in  the 
gray  or  the  white  substance.  Fatty  acids  and  neutral  fats  maybe  prepared 
from  the  brain  and  nerves;  but  as  these  may  be  readily  derived  from  a 
decomposition  of  lecithin  and  protagon.  which  exist  in  the  fatty  tissue 
between  the  nerve-axes,  it  is  difficult  to  decide  what  part  the  fatty  acids 
and  neutral  fats  play  as  constituents  of  the  real  nerve-substance.  Choles- 
terin  is  also  found  in  the  brain  and  nerves,  in  part  free  and  in  part  in  a  chem- 
ical combination  of  unknown  constitution  (Baumstark).  Cholesterin  seems 
to  occur  in  greater  abundance  in  the  white  substance.  Besides  these  sub- 
stances the  nerve  tissue,  especially  the  white  substance,  contains  doubtless 
a  number  of  other  constituents  not  well  known,  and  among  which  are 
several  containing  phosphorus.  Thudichum,8  who  has  made  thorough 
investigations  of  the  brain  and  has  described  a  great  number  of  brain  con- 
stituents, has  given  the  name  phosphatides  to  all  substances  of  the  brain 
containing  the  phosphoric-acid  radical.  Those  phosphatides,  which  con- 
tain only  one  phosphoric-acid  radical,  are  called  monophosphatides,  those 
with  two  such  radicals,  diphosphatides.  The  monophosphatides  can  con- 
tain one.  two,  or  more  nitrogen  atoms  in  their  molecule,  while  there  are  also 
nitrogen-free  monophosphatides.  Irrespective  of  the  relation  between 
phosphorus  and  nitrogen  certain  phosphatides  differ  from  the  lecithins  by 
not  yielding  any  glycerophosphoric  acid.  These  investigations  of  Thudi- 
chum are  without  doubt  of  great  importance,  but  as  they  have  not  been 
repeated  we  cannot  enter  into  a  discussion  of  the  bodies  described  by  him. 

By  allowing  water  to  act  on  the  contents  of  the  medulla,  round  or 

1  Pfliiger's  Arch.,  13. 

2  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  26;  Baumstark,  Zeitschr.  f .  physiol. 
Chem.,  9. 

3  Thudichum,  Die  chemische  Konstitution  des  Gehirns  des  Menschen  und  der  Tiere. 
Tubingen,  1901. 


408  BRAIN  AND  NERVES. 

oblong  double-contoured  drops  or  fibres,  not  unlike  double-contoured 
nerves,  are  formed.  This  remarkable  formation,  which  can  also  be  seen  in 
the  medulla  of  the  dead  nerve,  has  been  called  "myeline  forms,"  and  they 
were  formerly  considered  as  produced  from  a  special  body,  "  myeline." 
Myeline  forms  may,  however,  be  obtained  from  other  bodies,  such  as  impure 
protagon,  lecithin,  fat,  and  impure  cholesterin,  and  they  depend  upon  a 
decomposition  of  the  constituents  of  the  medulla.  According  to  Gad  and 
Heymans  i  myeline  is  lecithin  in  a  free  condition  or  in  loose  chemical  com- 
bination. 

The  extractive  bodies  seem  to  be  almost  the  same  as  in  the  muscles. 
One  finds  creatine,  which  may,  however,  be  absent  (B aumstark)  ,  xanthine 
bodies,  inosite,  lactic  acid  (also  fermentation  lactic  acid) ,  phosphocarnic  acid, 
uric  acid,  jecorin  (according  to  Baldi,2  in  the  human  brain),  and  the  diamine 
neuridine,  C5H14N2,  discovered  by  Brieger  3  and  which  is  most  interesting 
because  of  its  appearance  in  the  putrefaction  of  animal  tissues  or  in  cultures 
of  the  typhoid  bacillus.  Under  pathological  conditions  leucin  and  urea  have 
been  found  in  the  brain.  Urea  is  also  a  physiological  constituent  of  the 
brain  of  cartilaginous  fishes. 

Of  the  above-mentioned  constituents  of  the  nerve-substance  protagon 
and  its  decomposition  products,  the  cerebrins  or  cerebrosides,  must  be 
specially  described. 

Protagon.  This  body,  which  was  discovered  by  Liebreich,  is  a  nitrog- 
enized  and  phosphorized  substance  whose  elementary  composition,  accord- 
ing to  Gamgee  and  Blankenhorn,  is  C  66.39,  H  10.69,  N  2.39,  and  P  1.068 
per  cent.  Baumstark  and  Ruppel  obtained  the  same  figures,  while  Lieb- 
reich found  an  average  of  2.80  per  cent  N  and  1.23  per  cent  P.  Kossel 
and  Freytag,  who  obtained  still  higher  figures  for  the  nitrogen,  namely, 
3.25  per  cent,  and  somewhat  lower  figures  for  the  phosphorus,  0.97  per  cent, 
found  some  sulphur,  an  average  of  0.51  per  cent,  regularly  in  the  protagon. 
Ruppel  also  found  some  sulphur,  but  in  such  small  quantity  that  he  con- 
sidered it  as  a  contamination.  On  boiling  with  baryta-water  protagon 
yields  the  decomposition  products  of  lecithin,  namely,  fatty  acids,  glycero- 
phosphoric  acid,  and  choline  (neurine?),  and  besides  this,  as  above  stated,  also 
cerebrin.  Kossel  and  Freytag  found  that  protagon  not  only  yielded  cere- 
brin  in  its  decomposition,  but  two  and  perhaps  indeed  three  cerebrosides 
(see  below),  namely,  cerebrin,  kerasin  (homocerebrin),  and  encephalin. 
Because  of  this  behavior,  and  also  because  of  the  varying  elementary  com- 
position although  the  greatest  care  was  taken  in  the  preparation,  Freytag 
considers  it  very  probable  that  there  are  several  protagons.     Recent  investi- 


1  Du  Bois-Reymond 's  Arch.,  1890. 

2 Ibid.,  1887,  Supplbd. 

1  Brieger,  Ueber  Ptomaine.     Berlin,  1885  and  1886. 


PRO  T  AGON.  409 

gations  of  Worner  and  Thierfelder,  as  well  as  those  of  Lesem  and  Gies,1 
show  that  protagon  is  not  a  unit  substance  but  a  mixture. 

On  boiling  with  dilute  mineral  acids,  protagon  yields  among  other  sub- 
stances a  reducing  carbohydrate.  On  oxidation  with  nitric  acid  protagon. 
yields  higher  fatty  acids. 

Protagon  appears,  when  dry,  as  a  loose  white  powder.  It  dissolves  in 
alcohol  of  85  vols,  per  cent  at  45°  C,  but  separates  on  cooling  as  a  snow- 
white,  flaky  precipitate,  consisting  of  balls  or  groups  of  fine  crystalline 
needles.  It  decomposes  on  heating  even  below  100°  C.  It  is  hardly  soluble 
in  cold  alcohol  or  ether,  but  dissolves  on  warming.  It  swells  in  little  water 
and  partly  decomposes.  With  more  water  it  swells  to  a  gelatinous  or  pasty 
mass,  which  with  much  water  yields  an  opalescent  liquid.  On  fusing  with 
saltpeter  and  soda,  alkali  phosphates  are  obtained. 

Protagon  is  prepared  in  the  following  way:  An  ox-brain  as  fresh  as 
possible,  with  the  blood  and  membranes  carefully  removed,  is  ground  fine 
and  then  extracted  for  several  hours  with  alcohol  of  85  vols,  per  cent  at 
45°  C,  filtered  at  the  same  temperature,  and  the  residue  extracted  with 
warm  alcohol  until  the  filtrate  does  not  yield  a  precipitate  at  0°  C.  The 
several  alcoholic  extracts  are  cooled  to  0°  C.  and  the  precipitates  united  and 
completely  extracted  with  cold  ether,  which  dissolves  the  cholesterin  and 
lecithin-like  bodies.  The  residue  is  now  strongly  pressed  between  filter- 
paper  and  allowed  to  dry  over  sulphuric  acid  or  phosphoric  anhydride.  It 
is  now  pulverized,  digested  with  alcohol  at  45°  C,  filtered,  and  slowly  cooled 
to  0°  C.  The  crystals  which  separate  may  be  purified  when  necessary  by 
recrystallization. 

The  same  steps  are  taken  when  one  wishes  to  detect  the  presence  of  pro- 
tagon. 

On  decomposing  protagon  or  the  protagons  by  the  gentle  action  of 
alkalies  we  obtain  as  cleavage  products,  as  above  stated,  one  or  more  bodies 
which  Thudichum  has  embraced  under  the  name  cerebrosides.  The  cere- 
brosides  are  nitrogenous  substances  free  from  phosphorus,  which  yield  a 
reducing  variety  of  sugar  (galactose)  on  boiling  with  dilute  mineral  acids. 
On  fusing  with  potash  or  by  oxidation  with  nitric  acid  they  yield  higher 
fatty  acids:  palmitic  or  etearic  acids.  The  cerebrosides  isolated  from  the 
brain  are  cerebrin,  kerasin,  encephalin,  and  cerebron.  The  bodies  isolated 
by  Kossel  and  Freytag  from  pus,  and  called  pyosin,  and  pyogenin  also 
belong  to  the  cerebrosides. 

Cerebrin.  Under  this  name  W.  Muller  2  first  described  a  nitrogenous 
substance,  free  from  phosphorus,  which  he  obtained  by  extracting  with  boil- 

1  Gamgee  and  Blankenhorn,  Zeitschr.  f.  physiol.  Chem.,  3;  Baumstark,  1.  c. ; 
Ruppel,  Zeitschr.  f.  Biologie,  31;  Liebreich,  Annal.  d.  Chem.  u.  Pharm.,  134;  Kossel 
and  Freytag,  Zeitschr.  f.  physiol.  Chem.,  17;  Worner  and  Thierfelder,  ibid.,  30;  Lesem 
and  Gies,  Amer.  Journ.  of  Physiol.,  8. 

2  Annal.  d.  Chem.  u.  Pharm.,  105. 


410  BRAIN  AND  NERVES. 

ing  alcohol  a  brain-mass,  which  had  been  previously  boiled  with  baryta- 
water.  Following  a  method  essentially  the  same,  but  differing  somewhat, 
Geoghegan  *  prepared  from  the  brain  a  cerebrin  with  the  same  properties 
as  aIuller's,  but  containing  less  nitrogen.  According  to  Parous  2  the 
cerebrin  isolated  by  Geoghegan,  as  well  as  by  Muller,  consists  of  a  mixture 
of  three  bodies,  ' '  cerebrin, "  "  homocerebrin, ' '  and  ' '  encephalin. ' '  Kossel 
and  Freytag  isolated  two  cerebrosides  from  protagon  which  were  identical 
with  the  cerebrin  and  homocerebrin  of  Parous.  According  to  these  inves- 
tigators the  two  bodies  phrenosin  and  kerasin,  as  described  by  Thudichum, 
seem  to  be  identical  with  cerebrin  and  homocerebrin. 

Cerebrin,  according  to  Parous,  has  the  following  composition:  C  69.08, 
H  11.47,  N  2.13,  0  17.32  per  cent,  which  corresponds  with  the  analyses  made 
by  Kossel  and  Freytag.  No  formula  has  been  given  to  this  body.  In 
the  dry  state  it  forms  a  pure  white,  odorless,  and  tasteless  powder.  On 
heating  it  melts,  decomposes  gradually,  smells  like  burnt  fat,  and  burns 
with  a  luminous  flame.  It  is  insoluble  in  water,  dilute  alkalies,  or  baryta- 
water;  also  in  cold  alcohol  and  in  cold  or  hot  ether.  On  the  contrary,  it  is 
soluble  in  boiling  alcohol  and  separates  as  a  flaky  precipitate  on  cooling, 
and  this  is  found  to  consist  of  a  mass  of  balls  or  grains  on  microscopical 
examination.  Cerebrin  forms  a  compound  with  baryta,  which  is  insoluble 
in  water,  and  is  decomposed  by  the  action  of  carbon  dioxide.  Cerebrin 
dissolves  in  concentrated  sulphuric  acid,  and  on  warming  the  solution  it 
becomes  blood-red.  The  variety  of  sugar  split  off  on  boiling  with  mineral 
acids — the  so-called  brain-sugar — is,  according  to  Thierfelder,3  galactose. 

Kerasin  (according  to  Thudiohum),  or  homocerebrin  (according  to 
Parous),  has  the  following  composition:  C  70.06,  H  11.60,  N  2.23,  and 
O  16.11  per  cent.  Encephalin  has  the  composition  C  68.40,  H  11.60, 
N  3.09,  and  O  16.91  per  cent.  Both  bodies  remain  in  the  mother-liquor 
after  the  impure  cerebrin  has  precipitated  from  the  warm  alcohol.  These 
bodies  have  the  tendency  of  separating  as  gelatinous  masses.  Kerasin  is 
similar  to  cerebrin,  but  dissolves  more  easily  in  warm  alcohol  and  also  in 
warm  ether.  It  may  be  obtained  as  extremely  fine  needles.  Encephalin 
is,  according  to  Parous,  a  transformation  product  of  cerebrin.  In  the 
perfectly  pure  state  it  crystallizes  in  small  lamellae.  It  swells  into  a  pasty 
mass  in  warm  water.  Like  cerebrin  and  kerasin,  it  yields  a  reducing  sub- 
stance (probably  galactose)  on  boiling  with  dilute  acid. 

The  cerebrins  are  generally  prepared  according  to  Muller 's  method. 
The  brain  is  first  stirred  with  baryta-water  until  it  appears  like  thin  milk, 
and  then  it  is  boiled.  The  insoluble  parts  are  removed,  pressed,  and  re- 
peatedly boiled  with  alcohol,  which   is  filtered  while  boiling  hot.     The 

1  Zeitschr.  f.  physiol.  Chem.,  3. 

5  Parcus,  Ueber  einige  neue  Gehirnstoffe.     Inaug.-Diss.  Leipzig,  1881. 

3  Zeitschr.  f .  physiol.  Chem. ,  14. 


KERASIN,  CEBEBRON,  AND  CEPIIALIN.  411 

impure  cerebrin  which  separates  on  cooling  is  freed  from  cholcsterin  and 
fat  by  means  of  ether  and  then  purified  by  repeated  solution  in  warm 
alcohol.  According  to  Parcuk  this  repeated  solution  in  alcohol  is  con- 
tinued until  ..no  gelatinous  separation  of  homocerebrin  or  encephalin  takes 
place. 

Acording  to  Geoghegan  's  method  the  brain  is  first  extracted  with  cold 
alcohol  and  ether  and  then  boiled  with  alcohol.  The  precipitate  which 
separates  on  the  cooling  of  the  alcoholic  filtrate  is  treated  with  ether  and 
then  boiled  with  baryta-water.  The  insoluble  residue  is  purified  by  repeated 
solution  in  boiling  alcohol. 

The  cerebrin  may  also  be  obtained  from  other  organs  by  employing  the 
above  methods.  The  quantitative  estimation,  when  such  is  desired,  may  be 
performed  in  the  same  way. 

KosSEL  and  Freytag  prepare  cerebrin  from  protagon  by  saponifying  it 
in  solution  in  methyl  alcohol  with  a  hot  solution  of  caustic  baryta  in 
methyl  alcohol.  The  precipitate  is  filtered  off  and  decomposed  in  water  by 
carbon  dioxide  and  the  cerebrin  or  cerebroside  extracted  from  the  insoluble 
residue  with  hot  alcohol. 

Cerebron  is  a  substance  belonging  to  the  cerebroside  group  which  can 
be  prepared  from  the  brain  without  saponification  with  baryta  even 
at  a  temperature  below  50°  C,  hence  it  may  exist  preformed  in  the 
brain.  This  substance  first  isolated  by  Worner  and  Thierfelder  l  has 
the  composition  C  69.16,  H  11.54,  N  1.76,  O  17.54  per  cent.  It  melts  at 
212°,  dissolves  in  warm  alcohol  and  separates  out  on  cooling.  From  proper 
solvents  (acetone  containing  chloroform)  it  may  be  separated  as  small 
needles  or  plates.  If  cerebron  is  suspended  in  85  per  cent  alcohol  at  a 
temperature  of  50°  C.  it  balls  together  in  amorphous  masses  and  from 
these  needle  and  leaf-shaped  crystals  gradually  form.  Cerebron  also 
yields  galactose. 

Cephalin  is  a  body  similar  to  lecithin,  whose  formula,  based  upon  the 
investigations  of  Thudichum  and  of  W.  Koch,  is  probably  C42H82NP013. 
Cephalin  contains  only  one  methyl  group  and  according  to  Koch  is  probably 
a  dioxystearylmonomethyl  lecithin.  It  is  amorphous  and  swells  up  in 
water  like  lecithin.  It  is  soluble  in  cold  ether,  glacial  acetic  acid,  and  chloro- 
form, but  is  insoluble  in  acetone  and  in  alcohol,  either  cold  or  warm.  It 
is  obtained  from  the  brain  after  dehydration  with  acetone  by  extraction 
with  ether  and  precipitating  the  concentrated  ethereal  extract  with  alcohol. 
The  cephalin  is  perhaps  identical  with  the  myeline  substance  isolated  by 
Zuelzer  2  from  the  brain.  According  to  Thudichum  it  contains  a  specially 
unsaturated  fatty  acid  called  cephalic  acid. 

Bethe  3  has  prepared  the  following  decomposition  products  from  the  brain 
of  the  horse  after  treatment  with  CuCl2  and  alkali:  aminocerebrinic-<icid  glucoside, 

1  Zeitschr.  f .  physiol.  Chem. ,  30. 

3  W.  Koch,  Zeitschr.  f.  physiol.  Chem.,  36;  Zuelzer,  arid.,  27. 

8  Arch.  f.  exp.  Path.  u.  Phann.,  4S. 


412  BRAIN  AND  NERVES. 

C44H8108N,  which  on  boiling  with  hydrochloric  acid  yields  cerebrinic  acid,  amino- 
cerebrinic-acid  chloride,  and  a  hexose  (galactose  ?) ;  phrenin,  perhaps  identical 
with  Thudichum's  krinosin;  cerebrinic-phosphoric  acid,  and  a  stearic  acid  differing 
somewhat  from  the  ordinary  one. 

N-'uridins,  C5HUN2,  is  a  non-poisonous  diamine  discovered  by  Brieger,  and 
which  was  obtained  by  him  in  the  putrefaction  of  meat  and  gelatine,  and  from 
cultures  of  the  typhoid  bacillus.  It  also  occurs  under  physiological  conditions 
in  the  brain,  and  as  traces  in  the  yolk  of  the  egg. 

Xeuridine  dissolves  in  water  and  yeilds  on  boiling  with  alkalies  a  mixture  of  - 
dimethylamine  and  trimethylamine.  It  dissolves  with  difficulty  in  amyl  alcohol. 
It  is  insoluble  in  ether  or  absolute  alcohol.  la  the  free  state  neuridine  has  a  peculiar 
odor,  suggesting  semen.  With  hydrochloric  acid  it  gives  a  combination  crystalliz- 
ing in  long  needles.  With  platinic  chloride  or  gold  chloride  it  gives  crystallizable 
double  combinations  which  are  valuable  in  its  preparation  and  detection. 

The  so-called  corpuscula  amylacea,  which  occur  on  the  upper  surface  of  the 
brain  and  in  the  pituitary  gland,  are  colored  more  or  less  pure  violet  by  iodine 
and  more  blue  by  sulphuric  acid  and  iodine.  They  consist,  perhaps,  of  the  same 
substance  as  certain  prostatic  calculi,  but  they  have  not  been  closely  investigated. 

Quantitative  Composition  of  the  Brain.  The  quantity  of  water  is  greater 
in  the  gray  than  in  the  white  substance,  and  greater  in  new-born  or  young 
individuals  than  in  adults.  The  brain  of  the  foetus  contains  879-926  p.  m. 
water.  According  to  the  observations  of  Weisbach  1  the  quantity  of 
water  in  the  several  parts  of  the  brain  (and  in  the  medulla)  varies  at  differ- 
ent ages.  The  following  figures  are  in  1000  parts — A  for  men  and  B  for 
women : 

20-30  Years.  30-50  Years.  50-70  Years.  70-94  Years. 

A.  B.  A.  B.  A.  B.  A.  B. 

White  substance  of  the 

brain  695.6  682.9  683.1  703.1  701.9  689.6  726.1  722.0 

Grav,  ditto 833.6  826.2  836.1  830.6  838.0  838.4  847.8  839.5 

Gyri  784.7  792.0  795.9  772.9  796.1  796.9  802.3  801.7 

Cerebellum 788.3  794.9  778.7  789.0  787.9  784.5  803.4  797.9 

Pons  Varolii 734.6  740.3  725.5  722.0  720.1  714.0  727.4  724.4 

Medulla  oblongata 744.3  740.7  732.5  729.8  722.4  730.6  736.2  733.7 

Quantitative  analyses  of  the  brain  have  also  been  made  by  Petrowsky  2 
of  ox-brain,  and  by  Baumstark  of  the  brain  of  a  horse.  In  the  analysis 
of  Petrowsky  the  protagon  has  not  been  considered,  and  all  organic,  phos- 
phorized  substances  were  calculated  as  lecithin.  On  these  grounds  these 
analyses  are  not  of  much  value  from  a  certain  standpoint.  In  Baumstark  's 
analyses  the  gray  and  the  white  substance  could  not  be  sufficiently  sepa- 
rated, and  these  analyses,  on  this  account,  show  partly  an  excess  of  white 
and  partly  an  excess  of  gray  substance;  nearly  one-half  of  the  organic 
bodies,  chiefly  consisting  of  bodies  soluble  in  ether,  could  not  be  exactly 
analyzed.  Neither  of  these  analyses  gives  sufficient  explanation  of  the 
quantitative  composition  of  the  brain. 

The  analyses  made  up  to  the  present  time  give,  as  above  stated,  an 

1  Cited  from  K.  B.  Hofmann's  Lehrb.  d.  Zoochemie  (Wien,  1876),  121. 

7  Pfluger's  Arch  ,  7 


COMPOSITION  OF   THE  BRAIN.  413 

unequal  division  of  the  organic  constituents  in  the  gray  and  white  sub- 
stance. In  the  analyses  of  Petrowsky  the  quantity  of  proteids  and  gela- 
tine-foxming  substances  in  the  gray  matter  was  somewhat  more  than  one 
half,  and  in  the  white  about  one  quarter  of  the  solid  organic  substances. 
The  quantity  of  cholesterin  in  the  white  was  about  one  half,  and  in  the 
gray  substance  about  one  fifth  of  the  solid  bodies.  A  greater  quantity  of 
soluble  salts  and  extractive  bodies  was  found  in  the  gray  substance  than 
in  the  white  (Baumstark).  The  following  analyses  of  Baumstark  give 
the  most  important  known  constituents  of  the  brain  calculated  in  1000 
parts  of  the  fresh,  moist  substance.  A  represents  chiefly  the  white,  and  B 
chiefly  the  gray  substance. 

A.  B 

Water 695.35  769.97 

Solids 304.65  230.03 

Protagon 25.11  10.80 

Insoluble  proteid  and  connective  tissue 50 .  02  60 .  79 

Cholesterin,  free 18 .  19  6 .  30 

combined 26.96  17.51 

Xuclein 2 .  94  1 .  99 

Neurokeratin 18 .  93  10 .  43 

Mineral  bodies 5 .  23  5 .  62 

The  remainder  of  the  solids  probably  consists  chiefly  of  lecithin  and 
other  phosphorizecl  bodies.  Of  the  total  amount  of  phosphorus  15-20 
p.  m.  belongs  to  the  nuclein,  50-60  p.  m.  to  the  protagon,  150-160  p.  m. 
to  the  ash,  and  770  p.  m.  to  the  lecithin  and  the  other  phosphorized  organic 
substances. 

According  to  Noll  the  white  substance  of  the  spinal  marrow  is  some- 
what richer  in  protagon  than  the  brain,  and  in  nerve  degeneration  the 
quantity  of  protagon  diminishes.  The  method  used  by  him  would  not 
allow  of  an  exact  determination  of  the  protagon.  Mott  and  Halliburton  l 
have  also  shown  that  in  degenerative  diseases  of  the  nervous  system  the 
quantity  of  substances  containing  phosphorus  dimini  hes  and  that  in  these 
cases,  especially  in  general  paralysis,  choline  passes  into  the  cerebrospinal 
fluid  and  the  blood.  Donath  2  has  been  able  to  prove  the  occurrence  of 
choline  in  the  cerebrospinal  fluid  in  ep  leptics  and  in  several  different  organic 
diseases  of  the  nervous  system  He  considers  the  choline  as  the  substance 
instrumental  in  causing  the  seizures.  In  degenerated  nerves  the  quantity 
of  water  increases  and    he  phosphorus  decreases. 

The  quantity  of  neurokeratin  in  the  nerves  and  in  the  different  parts  of 
the  b  ain  has  been  carefully  determined  by  Kuhne  and  Chittenden.3 
They  found  3.16  p.  m.  in  the  plexus  brachialis,  3.12  p.  m.  in  the  cortex  of 


'Noll,  Zeitschr.  f.  physiol.  Chem.,  27;  Mott  and  Halliburton,  Philos.  Transact., 
Ser.  B,  191  (1899)  and  194  (1901). 
7  Zeitschr.  f    physiol.  Chem.,  39. 
3  Zeitschr.  f.  Biologie,  26. 


414  BRAIN  AND  NERVES. 

the  cerebellum,  22.434  p.  m.  in  the  white  substance  of  the  cerebrum,  25.72- 
29.02  p.  m.  in  the  white  substance  of  the  corpus  callosum,  and  3.27  p.  m. 
in  the  gray  substance  of  the  cortex  of  the  cerebrum  (when  free  as  possible 
from  white  substance).  The  white  is  decidedly  richer  in  neurokeratin  than 
the  peripheral  nerves  or  the  gray  substance.  According  to  Griffiths  x 
neurochitin  replaces  neurokeratin  in  insects  and  Crustacea,  the  quantity  of 
the  first  being  10.6-12  p.  m. 

The  quantity  of  mineral  constituents  in  the  brain  amounts  to  2.95-7.08 
p.  m.  according  to  Geoghegan.  He  found  in  1000  parts  of  the  fresh, 
moist  brain  0.43-1.32,  CI;  0.956-2.016,  P04;j  0.244-0.796,  C03;  0.102- 
0.220,  S04;  0.01-0.098,  Fe2(P04)2;  0.005-0.022,  Ca;  0.016-0.072,  Mg;  0.58- 
1.778,  K;  0.450-1.114,  Na.  The  gray  substance  yields  an  alkaline  ash,  the 
white  an  acid  ash. 

Appendix. 

The  Tissues  and  Fluids  of  the  Eye. 

The  retina  contains  in  all  865-899.9  p.  m.  water,  57.1-84.5  p.  m. 
proteid  bodies — myosin,  albumin,  and  mucin  (?),  9.5-28.9  p.  m.  lecithin, 
and  8.2-11.2  p.  m.  salts  (Hoppe-Seyler  and  Cahn  2).  The  mineral  bodies 
consist  of  422  p.  m.  Na,HP04  and  352  p.  m.  NaCl. 

Those  bodies  which  form  the  different  segments  of  the  rods  and  cones 
have  not  been  closely  studied,  and  the  greatest  interest  is  therefore  con- 
nected with  the  coloring-matters  of  the  retina. 

Visual  purple,  also  called  rhodopsin,  erythropsin,  or  visual  red,  is  the 
pigment  of  the  rods.  Boll  3  observed  in  1876  that  the  layer  of  rods  in  the 
retina  during  life  had  a  purplish-red  color  which  was  bleached  by  the  action 
of  light.  Kuhne  4  showed  later  that  this  red  color  might  remain  for  a  long 
time  after  the  death  of  the  animal  if  the  eye  was  protected  from  daylight 
or  investigated  by  a  sodium  light.  Under  these  conditions  it  was  also 
possible  to  isolate  and  closely  study  this  substance. 

Visual  red  (Boll)  or  visual  purple  (Kuhne)  has  become  known  mainly 
by  the  investigations  of  Kuhne.  The  pigment  occurs  chiefly  in  the  rods 
and  only  in  their  outer  parts.  In  animals  whose  retina  has  no  rods  the 
visual  purple  is  absent,  and  is  also  necessarily  absent  in  the  macula  lutea. 
In  a  variety  of  bat  (rhinolophus  Tiipposideros) ,  in  hens,  pigeons,  and  new- 
born rabbits,  no  visual  purple  has  been  found  in  the  rods. 

1  Compt.  rend.,  115. 

2  Zeitschr.  f.  physiol.  Chem.,  5. 

3  Monatsschr.  d.  Berl.  Akad.,  12.  Nov.,  187f3. 

*  The  investigations  of  Kuhne  and  his  pupils,  Ewald  and  Ayres,  on  the  visual  purple 
will  be  found  in  "  Untersuchungen  aus  dem  physiol.  Institut  der  Universitat  Heidel- 
berg," 1  and  2,  and  in  Zeitschr.  f.  Biologie,  32. 


PIGMENTS  OF  THE  EYE.  U5 

A  solution  of  visual  purple  in  water  which  contains  2-5  per  cent  crys- 
tallized bile,  which  is  the  best  solvent  for  it,  is  purple-red  in  color,  quite 
clear,  and  not  fluorescent.  On  evaporating  this  solution  in  vacuo  we 
obtain  a  residue  similar  to  ammonium  carminate  which  contains  violet  or 
black  grains.  If  the  above  solution  is  dialyzed  with  water,  the  bile  diffuses 
and  the  visual  purple  separates  as  a  violet  mass.  Under  all  circumsta 
even  when  still  in  the  retina,  the  visual  purple  is  quickly  bleached  by  direct 
sunlight,  and  with  diffused  light  with  a  rapidity  corresponding  to  the  in- 
tensity of  the  light.  It  passes  from  red  and  orange  to  yellow.  Red  light 
bleaches  the  visual  purple  slowly;  the  ultra-red  light  does  not  bleach  it  at 
all.  A  solution  of  visual  purple  hows  no  special  absorption-band.-.  In  it 
only  a  general  absorption  which  extends  from  the  red  side,  beginning  at 
D,  and  extending  to  the  G  line.      The  strongest  absorption  is  found  at  E. 

KoBTTGEN  and  Abelsdorf  l  have  shown  that  there  are,  in  ac  ordance  with 
KL'hxk's  views,  two  varieties  of  visual  purple,  the  one  occurring  in  mammals, 
turds,  and  amphibians,  and  the  other,  which  is  more  violet-red,  in  fishes.  The 
first  has  its  maximum  absorption  in  the  green  and  the  other  in  the  yellowish 
green. 

Visual  purple  when  heated  to  52-53°  C.  is  destroyed  after  several  hours, 
and  almost  instantly  when  heated  to  76°  C.  It  is  also  destroyed  by- 
alkalies,  acids,  alcohol,  ether,  and  chloroform.  On  the  contrary,  it  resists 
the  action  of  ammonia  or  alum  solution. 

As  the  visual  purple  is  easily  destroyed  by  light,  it  must  therefore  also 
be  regenerated  during  life.  Kuhne  has  also  found  that  the  retina  of  the 
eye  of  the  frog  becomes  bleached  when  exposed  for  a  long  time  to  strong 
sunlight,  and  that  its  color  gradually  returns  when  the  animal  is  placed  in 
the  dark.  This  regeneration  of  the  visual  purple  is  a  function  of  the  living 
cells  in  the  layer  of  the  pigment-epithelium  of  the  retina.  This  may  be 
inferred  from  the  fact  that  a  detached  piece  of  the  retina  which  has  been 
bleached  by  light  may  have  its  visual  purple  restored  if  it  is  carefully  laid 
on  the  choroid  having  layers  of  the  pigment-epithelium  attached.  The 
regeneration  has,  it  seems,  nothing  to  do  with  the  dark  pigment,  the 
melanin  or  fuscin,  in  the  epithelium-cells.  A  partial  regeneration  seems, 
according  to  Kuhne,  to  be  possible  in  the  retina  which  has  been  completely 
removed.  On  account  of  this  property  of  the  visual  purple  of  being  bleached 
by  light  during  life  we  may,  as  Kuhne  has  shown,  under  special  conditions 
and  by  observing  special  precautions,  obtain  after  death,  by  the  action  of 
intense  light  or  more  continuous  light,  the  picture  of  bright  object-,  such 
as  windows  and  the  like — so-called  optograms. 

The  physiological  importance  of  visual  purple  is  unknown.  It  follows 
that  the  visual  purple  is  not  essential  to  sight,  since  it  is  absent  in  certain 
animals  and  also  in  the  cones. 

'  Centralbl.  f   Physiol.,  9;   also  Maly's  Jahrosber.,  26,  351. 


416  BRAIN  AND  NERVES. 

Visual  purple  must  always  be  prepared  exclusively  in  a  sodium  light. 
It  is  extracted  from  the  net  membrane  by  means  of  a  watery  solution  of 
crystallized  bile.  The  filtered  solution  is  evaporated  in  vacuo  or  dialyzed 
until  the  visual  purple  is  separated.  To  prepare  a  visual-purple  solution 
perfectly  free  from  haemoglobin  the  solution  of  visual  purple  in  cholates  is 
precipitated  by  saturating  with  magnesium  sulphate,  washing  the  precipi- 
tate with  a  saturated  solution  of  magnesium  sulphate,  and  then  dissolving 
in  water  by  the  aid  of  the  cholates  simultaneously  precipitated.1 

The  Pigments  of  the  Cones.  In  the  inner  segments  of  the  cones  of  birds,  rep- 
tiles, and  fishes  a  small  fat-globule  of  varying  color  is  found.  Kuhne  2  has 
isolated  from  this  fat  a  green,  a  yellow,  and  a  red  pigment  called  respectively 
chlorophan,  xanthophan,  and  rhodophan. 

The  dark  pigment  of  the  epithelium-cells  of  the  net  membrane,  which  was 
formerly  called  melanin,  but  has  since  been  named  fuscin  by  Kuhne  and  May,3 
contains  iron,  dissolves  in  concentrated  caustic  alkalies  or  concentrated  sulphuric 
acid  on  warming,  but,  like  the  melanins  in  general  (see  Chapter  XVI) ,  has  been 
little  studied.  The  pigment  occurring  in  the  pigment-cells  of  the  choroid  seems 
to  be  identical  with  the  fuscin  of  the  retina. 

The  vitreous  humor  is  often  considered  as  a  variety  of  gelatinous  tissue. 
The  membrane  consists,  according  to  C.  Morner,  of  a  gelatine-forming 
substance.  The  fluid  contains  a  little  proteid  and  a  mucoid,  hyalomucoid, 
which  was  first  shown  by  Morner,  and  which  is  precipitated  by  acetic  acid. 
This  contains  12.27  per  cent  N  and  1.19  per  cent  S.  Among  the  extractives 
we  find  a  little  urea — according  to  Picard  5  p.  m.,  according  to  Rahlmann 
0.64  p.  m.  Pautz  4  found  besides  some  urea  paralactic  acid,  and,  in  con- 
firmation of  the  statements  of  Chabbas,  Jesner,  and  Ktjhn,  also  glucose 
in  the  vitreous  humor  of  oxen.  The  reaction  of  the  vitreous  humor  is  alka- 
line, and  the  quantity  of  solids  amounts  to  about  9-11  p.  m.  The  quantity 
of  mineral  bodies  is  about  6-9  p.  m.  and  the  proteids  0.7  p.  m.  In  regard 
to  the  aqueous  humor  see  page  224. 

The  Crystalline  Lens.  That  substance  which  forms  the  capsule  of  the 
lens  has  been  investigated  by  C.  Morner.  It  belongs,  according  to  him, 
to  a  special  group  of  proteins,  called  membranins.  The  membranin  bodies 
are  insoluble  at  the  ordinary  temperature  in  water,  salt  solutions,  dilute 
acids,  and  alkalies,  and,  like  the  mucins,  yield  a  reducing  substance  on 
boiling  with  dilute  mineral  acids.  They  contain  lead-blackening  sulphur. 
The  membranins  are  colored  a  very  beautiful  red  by  Millon  's  reagent,  but 
give  no  characteristic  reaction  with  concentrated  hydrochloric  acid  or  Adam- 

1  Kiihne,  Zeitschr.  f.  Biologie,  32. 

2  Kiihne,  Die  nichtbestiindigen  Farben  der  Netzhaut.  Untersuch.  aus  dem  physiol. 
Institut  Heidelberg,  1,  341. 

3  Kiihne,  ibid.,  2,324. 

4  Morner,  Zeitschr.  f.  physiol.  Chem.,  18;  Picard,  cited  from  Gamgee,  Physiol. 
Chem.,  1,  454;  Rahlmann,  Maly's  Jahresber.,  6;  Pautz,  Zeitschr.  f.  Biologie,  31.  A 
complete  review  of  the  literature  will  also  be  found  here. 


THE  CRYSTALLINE  LENS.  417 

kiewicz's  reagent.  They  arc  dissolved  with  greal  difficulty  by  pepsin- 
hydrochloric  acid  or  trypsin  solution,  but  are  soluble  in  dilute  acids  and 
alkalies  in  the  warmth.  Membranin  of  the  capsule  of  the  lens  contains 
1  1.10  per  cent  N  and  0.83  per  cent  S,  and  is  B  little  less  soluble  than  that 
from  Descemet's  membrane. 

The  chief  mass  of  the  solids  of  the  crystalline  lens  consists  of  proteids, 
whose  nature  has  been  investigated  by  C.  Moknkk.1  Some  of  these  pro- 
teids dissolve  in  dilute  salt  solution  while  others  remain  insoluble  in  the 
reagent. 

The  Insoluble  Proteid.  The  lens-fibres  consist  of  a  proteid  substance 
which  is  insoluble  in  water  and  in  salt  solution  and  to  which  Morn  BR  has  given 
the  name  albumoid.  It  dissolves  readily  in  very  dilute  acids  or  alkalies. 
Its  solution  in  caustic  potash  of  0.1  per  cent  is  very  similar  to  an  alkali- 
albuminate  solution,  but  coagulates  at  about  50°  C.  on  nearly  complete  neu- 
tralization and  the  addition  of  8  per  cent  NaCl.  Albumoid  has  the  following 
composition:  C  53.12,  H  6.8,  N  16.62,  and  S  0.79  per  cent.  The  lens-fibres 
themselves  contain  16.61  per  cent  N  and  0.77  per  cent  S.  The  inner  parts 
of  the  lens  are  considerably  richer  in  albumoid  than  the  outer.  The  quan- 
tity of  albumoid  in  the  entire  lens  amounts  on  an  average  to  about  48  per 
cent  of  the  total  weight  of  the  proteids  of  the  lens. 

The  Soluble  Proteid  consists,  exclusive  of  a  very  small  quantity  of 
albumin,  of  two  globulins,  a-  and  ft-crystallin.  These  two  globulins 
differ  from  each  other  in  this  manner:  a-crystallin  contains  16.68  per  cent 
N  and  0.56  per  cent  S;  /9-crystallin,  on  the  contrary,  17.04  per  cent  N  and 
1 .27  per  cent  S.  The  first  coagulates  at  about  72°  C.  and  the  other  at  63° 
C.  Besides  this, /9-crystallin  is  precipitated  from  a  salt-free  solution  with 
greater  difficulty  and  less  completely  by  acetic  acid  or  carbon  dioxide. 
These  globulins  are  not  precipitated  by  an  excess  of  NaCl  at  either  the  ordi- 
nary temperature  or  30°  C.  Magnesium  or  sodium  sulphate  in  substance 
precipitates  both  globulins,  on  the  contrary,  at  30°  C.  These  two  globulins 
are  not  equally  divided  in  the  mass  of  the  lens.  The  quantity  of  a-crystallin 
diminishes  in  the  lens  from  without  inwards;  /?-crystallin,  on  the  contrary. 
from  within  outwards. 

A.  Bechamp  distinguishes  the  two  following  proteid  bodies  in  the  watery 
extract  of  the  crystalline  lens:  phacozymase,  which  coagulates  at  55°  C.  and 
contains  a  diastatic  enzyme,  and  has  a  specific  rotatory  power  of  (a)/=  —41°, 
and  the  crystctfbumin,  with  a  specific  rotatory  power  of  (a)j=—  80°.3.  From 
the  residue  of  the  lens,  which  was  insoluble  in  water,  Bechamp  extracted,  by 
means  of  hydrochloric  acid,  a  proteid  body  having  a  specific  rotatory  power  of 
(a)]  =  -S0°.2,  which  he  called  crystalfibrin. 

The  lens  does  not  seem  to  contain  any  proteid  bodies  which  coagulate 
spontaneously  like  fibrinogen.     That  cloudiness  which  appears  after  death 

1  Zeitschr.  f.  physiol.  Chem.,  IS.     This  contains  also  the  pertinent  literature. 


418  BRAIN  AND  NERVES. 

depends,  according  to  Kuhne,  upon  the  unequal  changing  of  the  concen- 
tration of  the  contents  of  the  lens-tubes.  This  change  is  produced  by  the 
altered  ratio  of  diffusion.  A  cloudiness  of  the  lens  may  also  be  produced  in 
life  by  a  rapid  removal  of  water,  as,  for  example,  when  a  frog  is  plunged 
into  a  salt  or  sugar  solution.  The  appearance  of  cloudiness  in  diabetes  has 
been  attributed  by  some  to  the  removal  of  water.  The  views  on  this  sub- 
ject are,  however,  contradictory. 

The  average  results  of  four  analyses  made  by  Laptschinsky  1  of  the 
lens  of  oxen  are  here  given,  calculated  in  parts  per  1000: 

Proteids 349 .3 

Lecithin 2.3 

Cholesterin 2.2 

Fat 2.9 

Soluble  salts 5.3 

Insoluble  salts 2.3 

In  cataract  the  amount  of  proteids  is  diminished  and  the  amount  of 
cholesterin  increased. 

The  quantity  of  the  different  proteids  in  the  fresh  moist  lens  of  oxen  is 
as  follows,  according  to  Morner  2 : 

Albumoid  (lens-fibres) 170  p.  m. 

/?-Crystallin 110    " 

a-Crystallin 68    " 

Albumin 2    " 

The  corneal  tissue  has  been  previously  considered  (page  364).  The 
sclerotic  has  not  been  closely  investigated,  and  the  choroid  coat  is  chiefly 
of  interest  because  of  the  coloring-matter  (melanin)  it  contains  (see  Chapter 
XVI). 

Tears  consist  of  a  water-clear,  alkaline  fluid  of  a  saltish  taste.  Accord- 
ing to  the  analyses  of  Lerch  3  they  contain  982  p.  m.  water,  18  p.  m.  solids, 
with  5  p.  m.  albumin,  and  13  p.  m.  NaCl. 

The  Fluids  of  the  Inner  Ear. 

The  perilymph  and  endolymph  are  alkaline  fluids  which,  besides  salts, 
contain — in  the  same  amounts  as  in  transudates — traces  of  proteid,  and  in 
certain  animals  (codfish)  also  mucin.  The  quantity  of  mucin  is  greater  in 
the  perilymph  than  in  the  endolymph. 

Otoliths  contain  745-795  p.  m.  inorganic  substance,  which  consists 
chiefly  of  crystallized  calcium  carbonate.  The  organic  substance  is  very 
similar  to  mucin. 

1  Pfliiger's  Arch.,  13. 

2L.  c. 

3  Cited  from  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,  401. 


CHAPTER  XIII. 
ORGANS  OF  GENERATION. 

(a)  Male  Generative  Secretions. 

The  testes  have  been  little  investigated  chemically.  We  find  in  the 
testes  of  animals  proteid  bodies  of  different  kinds — seralbumin,  alkali  albu- 
minate (?),  and  an  albuminous  body  related  to  Rovidas'  hyaline  substance; 
also  leucin,  tyrosin,  creatine,  xanthine  bodies,  cholesterin,  lecithin,  inosite,  and 
fat.  In  regard  to  the  occurrence  of  glycogen  the  statements  are  somewhat 
contradictory.  Dareste  '  found  in  the  testes  of  birds  starch-like  granules, 
which  were  colored  blue  with  difficulty  by  iodine. 

The  semen  as  ejected  is  a  white  or  whitish-yellow,  viscous,  sticky  fluid 
of  a  milky  appearance,  with  whitish,  non-transparent  lumps.  The  milky 
appearance  is  due  to  spermatozoa.  Semen  is  heavier  than  water,  contains 
proteids,  has  a  neutral  or  faintly  alkaline  reaction  and  a  peculiar  specific 
odor.  Soon  after  ejection  semen  becomes  gelatinous,  as  if  it  were  coagu- 
lated, but  afterwards  becomes  more  fluid.  When  diluted  with  water  white 
(lakes  or  shreds  separate  (Hexle's  fibrin).  According  to  the  analyses  of 
Slowtzoff,2  human  semen  contains  on  an  average  96. S  p.  m.  solids  with 
9  p.  m.  inorganic  and  87.8  p.  m.  organic  substance.  The  amount  of  pro- 
tein substances  was  on  an  average,  22.6  p.  m.  and  1.69  p.  m.  bodies  soluble 
in  ether.  The  protein  substances  consist  of  nucleoproteids,  traces  of  mucin, 
albumin,  and  a  substance  similar  to  proteose  (found  earlier  by  Posxer). 
According  to  Cavazzani  3  semen  contains  relatively  considerable  nucleon. 
The  mineral  bodies  consist  chiefly  of  calcium  phosphate  and  rather  con- 
siderable NaCl.     Potassium  occurs  only  in  smaller  amounts. 

The  semen  in  the  vas  deferens  differs  chiefly  from  the  ejected  semen  in 
that  it  is  without  the  peculiar  odor.  This  last  depends  on  the  admixture 
with  the  secretion  of  the  prostate.  This  secretion,  according  to  Iversen, 
ha-  a  milky  appearance  and  ordinarily  an  alkaline  reaction,  very  rarely  a 
neutral  one,  and  contains  :  mall  amounts  of  proteids,  especially  nucleopro- 

1  Compt.  rend.,  74. 

2  Zeitschr.  f.  physiol.  Chem.,  35. 

3  Posner,  Berl.  klin.  Wochenschr.,  1888,  Xo.  21,  and  Centralbl.  f.  d  med.  Wissensch., 
1S0O;   Cavazzani,  Biochem.  Centralbl  ,  1,  502. 

419 


420  ORGANS  OF  GENERATION. 

teids,  besides  fibrinogen  and  a  substance  similar  to  mucin  (Stern  *),  and 
mineral  bodies,  especially  NaCl.  Besides  this  it  contains  an  enzyme  vesic- 
ulate (see  below),  lecithin,  choline  (Stern),  and  a  crystalline  combination 
of  phosphoric  acid  with  a  base,  C2H5N.  This  combination  has  been  called 
Bottcher's  spermine  crystals,  and  it  is  claimed  that  the  specific  odor  of  the 
semen  is  due  to  a  partial  decomposition  of  these  crystals. 

The  crystals  which  appear  on  slowly  evaporating  the  semen,  and  which 
are  also  observed  in  anatomical  preparations  kept  in  alcohol,  are  not  iden- 
tical with  the  Charcot-Leyden's  crystals  found  in  the  blood  and  in  the 
lymphatic  glands  in  leucaemia  (Th.  Cohn,  B.  Levy  2).  They  are,  according 
to  Schreiner,3  as  above  stated,  a  combination  of  phosphoric  acid  with  a 
base,  spermine,  C2H5N,  which  he  discovered. 

Spermine.  The  views  in  regard  to  the  nature  of  this  base  are  not  unanimous. 
According  to  the  investigations  of  Ladenburg  and  Abel,  it  is  not  improbable 
that  spermine  is  identical  with  ethylenimine ;  but  this  identity  is  disputed  by 
Majert  and  A.  Schmidt,  and  also  by  Poehl.  The  compound  of  spermine  with 
phosphoric  acid — Bottcher's  spermine  crystals — is  insoluble  in  alcohol,  ether, 
and  chloroform,  soluble  with  difficulty  in  cold  water,  but  more  readily  in  hot 
water,  and  easily  soluble  in  dilute  acids  or  alkalies,  also  alkali  carbonates  and 
ammonia.  The  base  is  precipitated  by  tannic  acid,  mercuric  chloride,  gold  chlo- 
ride, platinic  chloride,  potassium-bismuth  iodide,  and  phosphotungstic  acid. 
Spermine  has  a  tonic  action,  and  according  to  Poehl  4  it  has  a  marked  action  on 
the  oxidation  processes  of  the  animal  body. 

On  the  addition  of  potassium  iodide  to  spermatozoa  characteristic  dark-brown 
or  bluish-black  crystals  are  obtained — Florence's  sperma  reaction — which  is 
considered  by  many  as  a  reaction  for  spermine.  According  to  Bocarius,5  this 
reaction  is  due  to  choline. 

Camus  and  Gley6  have  found  that  the  prostate  fluid  in  certain  rodents 
has  the  property  of  coagulating  the  contents  of  the  seminal  vesicles.  This  prop- 
erty is  due  to  a  special  ferment  substance  (vesiculase)  of  the  prostate  fluid. 

The  spermatozoa  show  a  great  resistance  to  chemical  reagents  n  general. 
They  do  not  dissolve  completely  in  concentrated  sulphuric  acid,  nitric  acid, 
acetic  acid,  nor  in  boiling-hot  soda  solutions.  They  are  soluble  in  a  boiling- 
hot  caustic-potash  solution.  They  resist  putrefaction,  and  after  drying  they 
may  be  obtained  again  in  their  original  form  by  moistening  them  with  a  1 
per  cent  common-salt  solution.     By  careful  heating  and  burning  to  an  ash 

1  Iversen,  Nord.  med.  Ark.,  6;  also  Maly's  Jahresber.,  4,  358;  Stern,  Biochem. 
Centralbl,  1,  748. 

2Th.  Cohn,  Centralbl.  f.  aUg.  Path.  u.  path.  Anat.,  10  (1899);  B.  Levy,  Centralbl. 
f.  d.  med.  Wissensch.,  1899,  479. 

3  Annal.  d.  Chem.  u.  Pharm.,  194. 

4  Ladenburg  and  Abel..  Ber.  d.  deutsch.  chem.  Gesellsch.,  21;  Majert  and  A.  Schmidt, 
ibid.,  24;  Poehl,  Compt.  rend.,  115,  Berlin,  klin.  Wochenschr.,  1891  and  1893,  Deutsch. 
med.  Wochenschr.,  1892  and  1895,  and  Zeitschr.  f.  klin.  Med.    1894. 

5  In  regard  to  Florence's  sperma  reaction,  see  Posner,  Berl.  klin.  Wochenschr., 
1897,  and  Richter,  Wien.  klin.  Wochenschr.,  1897;  Bocarius,  Zeitschr.  f.  physiol. 
Chem.,  34. 

6  Compt.  rend,  de  Soc.  biolog.,  48,  49. 


SPERMATOZOA.  421 

the  shape  of  the  spermatozoa  may  be  seen  in  the  ash.     The  quantity  of 
ash  is  about  50  p.  m.  and  consists  mainly  (f)  of  potassium  phosphate. 

The  spermatozoa  show  well-known  movements,  but  the  cause  of  this  is 
not  known.  This  movement  may  continue  for  a  very  long  time,  as  under 
some  conditions  it  may  be  observed  for  several  days  in  the  body  after  death, 
and  in  the  secretion  of  the  uterus  longer  than  a  week.  Acid  liquids  stop 
these  movements  immediately;  they  are  also  destroyed  by  strong  alkalies, 
especially  ammoniacal  liquids,  also  by  distilled  water,  alcohol,  ether,  etc. 
The  movements  continue  for  a  longer  time  in  faintly  alkaline  liquids, 
especially  in  alkaline  animal  secretions,  and  also  in  properly  diluted  neu- 
tral salt  solutions. 

Spermatozoa  are  nucleus  formations  and  hence  are  rich  in  nucleic  acid, 
which  exists  in  the  heads.  The  tails  contain  proteid  and  are  besides  this 
rich  in  lecithin,  cholesterin,  and  fat,  which  bodies  only  occur  to  a  small 
extent  (if  at  all)  in  the  heads.  The  tails  seem  by  their  composition  to  be 
closely  allied  to  the  non-medullated  nerves  or  the  axis-cylinders.  In  the 
various  kinds  of  animals  investigated,  the  head  contains  nucleic  acid,  which 
in  fishes  is  partly  combined  with  protamins  and  partly  with  histons.  In 
other  animals,  such  as  the  bull  and  boar,  proteid-like  substances  occur  with 
the  nucleic  acid,  but  no  protamin. 

Our  knowledge  of  the  chemical  composition  of  spermatozoa  has  been 
greatly  enhanced  by  the  important  investigations  of  Miescher  *  on  salmon 
roe.  The  intermediate  fluid  of  the  spermatozoa  of  Rhine  salmon  is  a  dilute 
salt  solution  containing  1.3-1.9  p.  m.  organic,  and  6.5-7.5  p.  m.  inorganic 
bodies.  The  last  consist  chiefly  of  sodium  chloride  and  carbonate,  besides 
some  potassium  chloride  and  sulphate.  The  fluid  only  contains  traces  of  pro- 
teid, but  no  peptone.  The  tails  consist  of  419  p.  m.  proteid,  318.3  p.  m.  leci- 
thin, and  262.7  p.  m.  cholesterin  and  fat.  The  heads  extracted  with  alcohol- 
ether  contain  on  an  average  960  p.  m.  protamin  nucleate,  which  neverthe- 
less is  not  uniform,  but  is  so  divided  that  the  outer  layers  consist  of  basic 
protamin  nucleate,  while  the  inner  lave  s,  on  the  contrary,  consist  of  acid 
protamin  nucleate.  Besides  the  protamin  nucleate  there  is  present  in  the 
heads,  although  to  a  very  slight  extent,  unknown  organic  substances.  The 
unripe  salmon  spermatozoa,  while  developing,  also  contain  nucleic  acid,  but 
no  protamin,  with  a  proteid  substance,  "albuminose,"  which  probably  i-  a 
step  in  the  formation  of  protamin.  According  to  Kossel  and  Mathews,3 
in  the  herring  as  in  the  salmon,  the  heads  of  the  spermatozoa  consist  of 
protamin  nucleate  but  no  free  proteid. 

Spermatin  is  a  name  which  has  been  given  to  a  constituent  similar  to  alkali 
albuminate,  but  it  has  not  been  closely  studied. 


'See  Miescher,  "Die  histochemischen  und  physiologischen  Arbeiten  von  Friedrich 
Miescher,  gesammelt  und  herausgegeben  von  seinen  Freunden. "     Leipzig,  1897. 
2  Zeitschr.  f.  physiol.  Chem.,  23. 


422  ORGANS  OF  GENERATION. 

Prostatic  concrements  are  of  two  kinds.  One  is  very  small,  generally  oval  in 
shape,  with  concentric  layers.  In  young  but  not  in  older  persons  they  are  colored 
blue  by  iodine  (Iversen  l).  The  other  kind  is  larger,  sometimes  the  size  of  the 
head  of  a  pin  and  consisting  chiefly  of  calcium  phosphate  (about  700  p.  m.),  with 
only  a  very  small  amount  (about  160  p.  m.)  organic  substance. 

(b)  Female  Generative  Organs. 

The  stroma  of  the  ovaries  is  of  little  interest  from  a  physiologico- 
chemical  standpoint,  and  the  most  important  constituents  of  the  ovaries,  the 
Graafian  follicles  with  the  ovum,  have  not  thus  far  been  the  subject  of  a 
careful  chemical  investigation.  The  fluid  in  the  follicles  (of  the  cow)  does 
not  contain,  as  has  been  stated,  the  peculiar  bodies,  paralbumin  or  metalbu- 
min,  which  are  found  in  certain  pathological  ovarial  fluids,  but  seems  to  be  a 
serous  liquid.  The  corpora  lutea  are  colored  yellow  by  an  amorphous  pig- 
ment called  lutein.  Besides  this  another  coloring-matter  sometimes  occurs 
which  is  not  soluble  in  alkali;  it  is  crystalline,  but  not  identical  with  bili- 
rubin or  hEematoidin;  but  it  may  be  identified  as  a  lutein  by  its  spectro- 
scopic behavior  (Piccolo  and  Lieben;  Kuhne  and  Ewald  2). 

The  cysts  often  occurring  in  the  ovaries  are  of  special  pathological 
interest,  and  these  may  have  essentially  different  contents,  depending  upon 
their  variety  and  origin. 

The  serous  cysts  (Hydrops  folliculorum  Graafii),  which  are  formed 
by  a  dilation  of  the  Graafian  follicles,  contain  a  serous  liquid  which  has  a 
specific  gravity  of  1.005-1.022.  A  specific  gravity  of  1.020  is  less  frequent. 
Generally  the  specific  gravity  is  lower,  1.005-1.014,  with  10-40  p.  m.  solids. 
As  far  as  is  known,  the  contents  of  these  cysts  do  not  essentially  differ  from 
other  serous  liquids. 

The  proliferous  cysts  (myxoid  cysts,  colloid  cysts),  which  are  devel- 
oped from  Pfluger  's  epithelium-tubes,  may  have  a  content  of  a  decidedly 
variable  composition. 

We  sometimes  find  in  small  cysts  a  semi-solid,  transparent,  or  somewhat 
cloudy  or  opalescent  mass  which  appears  like  solidified  glue  or  quivering 
jelly,  and  which  has  been  called  colloid  because  of  its  physical  properties. 
In  other  cases  the  cysts  contain  a  thick,  tough  mass  which  can  be  drawn  out 
into  long  threads,  and  as  this  mass  in  the  different  cysts  is  more  or  less 
diluted  with  serous  liquids  their  contents  may  have  a  variable  consistency. 
In  still  other  cases  the  small  cysts  may  also  contain  a  thin,  watery  fluid. 
The  color  of  the  contents  is  also  variable.  Sometimes  they  are  bluish- 
white,  opalescent,  and  again  they  are  yellow,  yellowish-brown,  or  yellowish 
with  a  shade  of  green.  They  are  often  colored  more  or  less  chocolate- 
brown  or  red-brown,  due  to  the  decomposed  blood-coloring  matters.  The 
reaction  is  alkaline  or  nearly  neutral.    The  specific  gravity,  which  may  vary 


1  Nord.  med.  Ark.,  6.  2  See  Chapter  VI,  page  181. 


COLLOID  AND  PSEUDOMUCIN. 

considerably,  is  generally  L.015-1.030,  but  may  occasionally  lie  L.005-1.010 

or   1.050-1.055.    The  amount  of  solids  is  very  variable.     In   rare  cases 
ii    amounts  to  only  10-20  p.  mv;  ordinarily  it  varies  between  50-70-100 

j).  in.     In  a  few  instances  150-200  ]>.  m.  solids  have  been  found. 

As  form-elements  one  finds  red  and  white  blood-corpuscles,  granular  cells, 
partly  fat-degenerated  epithelium  and  partly  large  so-called  Gluge's  cor- 
puscles, fine  granular  masses,  epithelium-cells,  cholesterin  crystals,  and  colloid 
corpuscles — large,  circular,  highly  refractive  formations. 

Though  the  contents  of  the  proliferous  cyst  may  have  a  variable  compo- 
sition, still  it  may  be  characterized  in  typical  cases  by  its  slimy  or  ropy 
consistency;  by  its  grayish-yellow,  chocolate-brown,  sometimes  whitish, 
gray  color;  and  by  its  relatively  high  specific  gravity,  1.015-1.025.  Such  a 
liquid  does  not  ordinarily  show  a  spontaneous  fibrin  coagulation. 

We  consider  colloid,  metalbumin,  and  paralbumin  as  characteristic  con- 
stituents of  these  cysts. 

Colloid.  This  name  does  not  designate  any  particular  chemical  sub- 
stance, but  is  given  to  the  contents  of  tumors  with  certain  physical  proper- 
ties similar  to  gelatine  jelly.  Colloid  is  found  as  a  pathological  product 
in  several  organs. 

Colloid  is  a  gelatinous  mass,  insoluble  in  water  and  acetic  acid;  it  is 
dissolved  by  alkalies  and  gives  a  liquid  which  is  not  precipitated  by  acetic 
acid  or  by  acetic  acid  and  potassium  ferrocyanide.  According  to  Ppannen- 
STIEL  '  such  a  colloid  is  designated  /^-pseudomucin.  Sometimes  a  colloid  is 
found  which,  when  treated  with  a  very  dilute  alkali,  gives  a  solution  similar 
to  a  mucin  solution.  Colloid  is  very  closely  related  to  mucin  and  is  con- 
sidered by  certain  investigators  as  a  modified  mucin.  An  ovarial  colloid 
analyzed  by  Panzer  contained  931  p.  m.  water,  57  p.  m.  organic  substance 
and  12  p.  m.  ash.  The  elementary  composition  was  C  47.27,  H  5.86,  N  8.40, 
S  0.79,  P  0.54,  and  ash  0.43  per  cent.  A  colloid  found  by  Wurtz  2  in  the 
hums  contained  C  48.09,  II  7.47,  N  7.00,  and  0(+S)  37.44  per  cent,  Col- 
loids of  different  origin  seem  to  be  of  varying  composition. 

Metalbumin.  This  name  Schereb  :!  gave  to  a  protein  substance  found 
by  him  in  an  ovarial  fluid.  The  metalbumin  was  considered  by  Scherer 
to  be  an  albuminous  body,  but  it  belongs  to  the  mucin  group,  and  it  is  for 
this  reason  culled  pseudomucin  by  IIammarstkx.4 

Pseudomucin.  This  body,  which,  like  the  mucins,  gives  a  reducing  sub- 
stance when  boiled  with  acids,  is  a  mucoid  of  the  following  composition: 

'Arch.  f.  Gynfik.,88. 

2  Panzer,  Zcitsehr.  f.  phvsiol.  Chem.,  2S;  Wurtz,  see  Lebert,  Beitr.  zur  Kenntniss 
des  Gallertkrebses,  Virchow's  Arch.,  4. 

3  Verh.  d.  physik.-med.  Gesellsch.  in  Wurzburg,  2,  and  Sitsungsber.  der  physik.- 
med.  Gesellsch.  in  Wurzbuig  fur  1864-1865;   Wiirzburg  med.  Zeitschr.,  7. 

4  Zeitschr.  f.  phvsiol.  Chem.,  6. 


424  ORGANS  OF  GENERATION. 

C  49.75,  H  6.98,  N  10.28,  S  1.25,  0  31.74  per  cent  (Hammarsten)  .  With 
water  pseudomucin  gives  a  slimy,  ropy  solution,  and  it  is  this  substance 
which  gives  the  fluid  contents  of  the  ovarial  cysts  their  typical  ropy  prop- 
erty. Its  solutions  do  not  coagulate  on  boiling,  but  only  become  milky 
or  opalescent.  Unlike  mucin,  pseudomucin  solutions  are  not  precipitated, 
by  acetic  acid.  With  alcohol  they  give  a  coarse  flocculent  or  thready 
precipitate  which  is  soluble  even  after  having  been  kept  under  water  or 
alcohol  for  a  long  time. 

Paralbumin  is  another  substance  discovered  by  Scherer,  and  which 
occurs  in  ovarial  liquids  and  also  in  ascitic  fluids  with  the  simultaneous 
presence  of  ovarial  cysts  and  rupture  of  the  same.  It  is  therefore  only  a 
mixture  of  pseudomucin  with  variable  amounts  of  proteid,  and  the  reactions 
of  paralbumin  are  correspondingly  variable. 

Mitjukoff  *  has  isolated  and  investigated  a  colloid  from  an  ovarial  cyst.  It 
had  the  following  composition:  C  51.76,  H  7.76,  N  10.7,  S  1.09,  and  O  28.69  per 
cent,  and  differed  from  mucin  and  pseudomucin  by  reducing  Fehling's  solution 
before  boiling  with  acid.  It  must  be  remarked  that  pseudomucin,  on  boiling 
sufficiently  long  with  alkali,  or  by  the  use  of  a  concentrated  solution  of  caustic 
alkali,  also  splits  and  causes  a  reduction.  This  reduction  is  nevertheless  weak 
as  compared  with  that  produced  after  boiling  with  an  acid.  The  body  isolated 
by  Mitjukoff  is  called  paramucin. 

The  pseudomucin  as  well  as  colloid  are  mucoid  substances  and  the 
carbohydrate  obtained  from  them  is  glucosamine  (chitosamine) ,  as  espe- 
cially shown  by  Fr.  Muller,  Neuberg  and  Heymann.2  From  pseudo- 
mucin Zangerle  3  obtained  30  per  cent  glucosamine  and  Neuberg  and 
Heymann  have  shown  that  the  glucosamine  is  the  only  carbohydrate 
regularly  taking  part  in  the  structure  of  these  substances.  Still  there  are 
also  statements  as  to  the  occurrence  of  chondroitin-sulphuric  acid  (or  an 
allied  acid)  in  pseudomucin  or  colloid  (Panzer),  but  this  is  not  constant 
according  to  the  experience  of  Hammarsten. 

The  detection  of  metalbumin  and  paralbumin  is  naturally  connected 
with  the  detection  of  pseudomucin.  A  typical  ovarial  fluid  containing 
pseudomucin  is,  as  a  ride,  sufficiently  characterized  by  its  physical  proper- 
ties, and  a  special  chemical  investigation  is  only  necessary  in  cases  where  a 
serous  fluid  contains  very  small  amounts  of  pseudomucin.  The  procedure 
is  as  follows:  The  proteid  is  removed  by  heating  to  boiling  with  the 
addition  of  acetic  acid ;  the  filtrate  is  strongly  concentrated  and  precipitated 
by  alcohol.  The  precipitate,  a  transformation  product  of  pseudomucin, 
is  carefully  washed  with  alcohol  and  then  dissolved  in  water.  A  part  of 
this  solution  is  digested  with  saliva  at  the  temperature  of  the  body  and  then 

1  K.  Mitjukoff,  Arch.  f.  Gynakol.,  49.     • 

2  Miiller,  Verh.  d.  Naturf.  Gesellsch.  in  Basel,  12,  part  2;  Neuberg  and  Heymann,. 
Hofmeister's  Beitrage,  2.     See  also  Leathes,  Arch.  f.  exp.  Path.  u.  Pharnx,  43. 

3  Munch,  med.  Wochenschr. ,  1900. 


CYSTS.  425 

tested  for  glucose  (derived  from  glycogen  or  dextrin).  If  glycogen  is  pres- 
ent, it  will  be  converted  into  glucose  by  the  saliva;  precipitate  again  with 
alcohol  and  then  proceed  as  in  the  absence  of  glycogen.  In  this  lust-men- 
tioned case,  first  add  acetic  acid  to  the  solution  of  the  alcohol  precipitate 
in  water  so  as  to  precipitate  any  existing  mucin.  The  precipitate  produced 
is  filtered  off,  the  filtrate  treated  with  2  percent  HC1  and  warmed  on  the 
water-bath  until  the  liquid  is  deep  brown  in  color.  In  the  presence  of 
pseudomucin  this  solution  gives  Trommer's  test. 

The  other  protein  bodies  which  have  been  found  in  cystic  fluids  are 
serglobulin  and  seralbumin,  peptone  (?),  mucin,  mucin-peptone  (?).  Fibrin 
occurs  only  in  exceptional  cases.  The  quantity  of  mineral  bodies  on  an 
average  amounts  to  about  10  p.  m.  The  amount  of  extractive  bodies 
(cholcsterin  and  urea)  and  fat  Is  ordinarily  2-4  p.  m.  The  remaining  solids, 
which  constitute  the  chief  mass,  are  albuminous  bodies  and  pseudomucin. 

The  intraligamentary,  papillary  cysts  contain  a  yellow,  yellowish-green, 
or  brownish-green  liquid  which  contains  either  no  pseudomucin  or  very 
little.  The  specific  gravity  is  generally  rather  high,  1.032-1.036,  with 
90-100  p.  m.  solids.  The  principal  constituents  are  the  simple  proteids  of 
blood-serum. 

The  rare  tubo-ovarial  cysts  contain  as  a  rule  a  watery,  serous  fluid  con- 
taining no  pseudomucin. 

The  parovarial  cysts  or  the  cysts  of  the  ligamenta  lata  may  attain  a 
considerable  size.  In  general,  and  when  quite  typical,  the  contents  are 
watery,  mostly  very  pale  yellow-colored,  water-clear  or  only  slightly  opal- 
escent liquids.  The  specific  gravity  is  low,  1.002-1.009,  and  the  solids  only 
amount  to  10-20  p.  m.  Pseudomucin  does  not  occur  as  a  typical  constit- 
uent; proteid  is  sometimes  absent,  and  when  it  does  occur  the  quantity  is 
very  small.  The  principal  part  of  the  solids  consists  of  salts  and  extractive 
bodies.  In  exceptional  cases  the  fluid  may  be  rich  in  proteid  and  may  show 
a  higher  specific  gravity. 

In  regard  to  the  quantitative  composition  of  the  fluid  from  ovarial  cysts 
we  refer  the  reader  to  the  work  of  Oerum.1 

E.  Ludwtg  and  R.  v.  Zeynek  2  have  recently  investigated  the  fat  from  dermoid 
cysts.  Besides  a  little  arachidic  acid,  they  found  oleic,  stearic,  palmitic,  and 
myristic  acids,  cetyl  alcohol,  and  a  cholesterin-like  substance. 

The  colloid  from  a  uterine  fibroma  analyzed  by  Stollmann3  contained  a 
pseudomucin  soluble  in  water  and  a  colloid  (paramucin)  insoluble  in  water,  both 
of  which  beh  ved  differently  with  alcohol  as  compared  to  the  corresponding  sub- 
stances from  ovarial  cysts. 


1  Kemiske  Studier  over  Ovnriecystevaedsker,   etc.     Koebenhavn,  1884.     See   also 
Maly's  Jahresber.,  14,  459. 

2  Zeitschr.  f.  physiol.  Chem.,  23. 

8  American  Gynecology,  March,  1903. 


428  ORGANS  OF  GENERATION. 

The  Ovum. 

The  small  ova  of  man  and  mammals  cannot,  for  evident  reasons,  be  the 
subject  of  a  searching  chemical  investigation.  Up  to  the  present  time  the 
eggs  of  birds,  amphibians,  and  fishes  have  been  investigated,  but  above  all 
the  hen's  egg.  We  will  here  occupy  ourselves  with  the  constituents  of  this 
last. 

The  Yolk  of  the  Hen's  Egg.  In  the  so-called  white  yolk,  which  forms 
the  germ  with  a  process  reaching  to  the  centre  of  the  yolk  (latebra),  and  form- 
ing a  layer  between  the  yolk  and  yolk-membrane,  there  occur  proteid,  nuclein, 
lecithin,  and  pota  sium  (Liebermann  *).  The  occurrence  of  glycogen  is 
doubtful.  The  yolk-membrane  consists  of  an  a  buminoid  similar  in  certain 
respects  to  keratin  (Liebermann)  . 

The  principal  part  of  the  yolk — the  nutritive  yolk  or  yellow — is  a 
viscous,  non-transparent,  pale-yellow  or  orange-}rellow  alkaline  emulsion 
of  a  mild  taste.  The  yolk  contains  vitellin,  lecithin,  cholesterin,  fat,  color- 
ing-matters, traces  of  neuridine  (Brieger2),  a  diastatic  enzyme  (Muller  and 
Masuyama),  purin  bases  (Mesernitzki  3),  glucose  in  very  small  quantities, 
and  mineral  bodies.  The  occurrence  of  cerebrin  and  of  granules  similar  to 
starch  (Dareste  4)  has  not  been  positively  proved. 

Ovovitellin.  This  body  which  is  generally  considered  as  a  globulin,  is  in 
reality  a  nucleoalbumin.  The  question  as  to  what  relationship  other  protein 
substances  which  are  related  to  ovovitellin,  like  the  aleuron-grains  of  cer- 
tain seeds  and  the  yolk  spherules  of  the  eggs  of  certain  fi=hes  and  amphib- 
ians, bear  to  this  substance  is  one  which  requires  further  investigation. 

The  ovovitellin  which  has  been  prepared  from  the  yolk  of  eggs  is  not  a 
pure  proteid  body,  but  always  contains  lecithin.  Hoppe-Seyler  found 
25  per  cent  lecithin  in  vitellin  and  also  some  pseudonuclein.  The  lecithin 
may  be  removed  by  boiling  alcohol,  but  the  vitellin  is  changed  thereby,  and 
it  is  the"efore  probable  hat  the  lecithin  is  chemically  united  with  the 
vitellin  (Hoppe-Seyler  5).  According  to  Osborne  and  Campbell,  the  so- 
called  ovovitellin  is  a  mixture  of  various  vitellin-lecithin  combinations,  with 
15-30  per  cent  of  lecith'n  The  proteid  substance  freed  from  lecithin  is  the 
■  in  all  these  compounds  and  has  the  following  composition:  C  51.24, 
II  7.16,  N  16.38,  S  1.04,  P  0.94,  0  23.24  per  cent.  These  figures  differ 
what  from. those  obtained  by  Gross  6  for  vitellin  prepared  by  another 

1  Pflii£er's  Arch..  43. 

2  Teber  Ptomaine.     Berlin,  1885. 

3  Miiller  and  Masuyama,  Zeitschr.  f.  Biologie,  39;  Mesernitzki,  Biochem.  Cen- 
tralbl..  1,  739. 

*  Compt.  rend.,  72. 
6  Med.  chem.  Untersuch.,  216. 

9  Osborne  and  Campbell,  Connecticut  Agric.  Exp.  Station,  23d  Ann.  Report,  New 
Haven,  1900;  Gross,  Zur  Kenntn.  d.  Ovovitellins,  Inaug.-Diss.  Strassburg,  1899. 


OVOVITELLIN  127 

method  (precipitation  with  (NHJaSOJ,  namely:  C  48.01,  H  6.35,  N  14.91- 

lf>.'.)7,  V  0.32 -0.35,  S  O.ss,  ami  the  composition  of  ovovitellin  is  therefore 
not  positively  known.  Gross  found  in  vitellin  a  globulin  coagulating  at 
76-77°  C.  in  a  solution  containing  hydrochloric  acid. 

( >n  the  pepsin  digestion  of  ovovitellin,  Osborne  and  Campbell  obtained 
a  pseudonuclein  with  varying  amounts  of  phosphorus,  2.52—1.1'.)  per  cent. 
BUNGE  '  prepared  a  pseudonuclein  by  digesting  the  yolk  with  gastric  juice, 
and  his  pseudonuclein,  according  to  him,  is  of  great  importance  in  the 
formation  of  he  blood,  and  on  these  grounds  he  called  it  hcematogen.  This 
hsematogen  has  the  following  composition:  C  42.11,  H  6.08,  N  14.73, 
S  0.55,  P  5.19,  Fe  0.29,  and  O  31.05  per  cent. 

Vitellin  is  similar  to  the  globulins  in  that  it  is  insoluble  in  water,  but 
on  the  contrary  soluble  in  dilute  neutral-salt  solutions  (although  the  solution 
is  not  quite  transparent).  It  is  also  soluble  in  hydrochloric  acid  of  1  p.  m. 
ami  in  very  dilute  solutions  of  alkalies  or  alkali  carbonates.  It  is  precipi- 
tated from  its  salt  solution  by  diluting  with  water,  and  when  allowed  to 
stand  some  time  in  contact  with  water  the  vitellin  is  gradually  changed, 
forming  a  substance  more  like  the  albuminates.  The  coagulation  tempera- 
ture for  the  solution  containing  salt  (NaCl)  lies  between  70°  and  75°  C, 
or.  when  heated  very  rapidly,  at  about  80°  C.  Vitellin  differs  from  the 
globulins  in  yielding  pseudonuclein  by  peptic  digestion.  It  is  not  always 
completely  precipitated  by  NaCl  in  substance.  The  ovovitellin  isolated  by 
Gross  gave  Molisch's  reaction.  Neuberg  2  has  also  split  off  glucosamine 
from  the  yolk  and  has  identified  it  as  norisosaccharic  acid.  It  is  difficult 
to  state  whether  this  glucosamine  was  derived  from  the  vitellin  or  from 
some  other  constituent  of  the  yolk. 

The  chief  points  in  the  preparation  of  ovovitellin  are  as  follows:  The 
yolk  is  thoroughly  agitated  with  ether;  the  residue  is  dissolved  in  a  10  per 
cent  common-salt  solution,  filtered,  and  the  vitellin  precipitated  by  adding 
an  abundance  of  water.  The  vitellin  is  now  purified  by  repeatedly  redis- 
solving  in  dilute  common-salt  solutions  and  precipitating  with  water. 

Ichthulin,  which  occurs  in  the  eggs  of  the  carp  and  other  fishes  is,  according 
to  Kossel  and  Walter,3  an  amorphous  modification  of  the  crystalline  body 
iclilhiilin.  which  occurs  in  the  eggs  of  the  carp.  Ichthulin  is  precipitated  on 
diluting  with  water.  It  used  to  be  considered  as  a  vitellin.  According  to  Walter 
it  yields  a  pseudonuclein  on  peptic  digestion;  and  this  pseudonuclein  gives  a 
reducing  carbohvdrate  on  boiling  with  sulphuric  acid.  Ichthulin  has  the  follow- 
ing composition!  C  53.42,  H  7.63,  X  15.63,  O  22.19,  S  0.41,  P  0.43.  It  also  con- 
tains iron.  The  ichthulin  investigated  by  Levene  from  codfish  epgs  had  the 
composition  C  52.44,  H  7.45,  X  15.96,  S  0.92,  P  0.65,  Fe+O  22.58  per  cent, 
yielded    no    reducing    substances   on   boiling    with   acids   and    behaved   similar 

1  Zeitschr.  f.  physiol.  Chem.,  9,  49. 

2  Ber.  d.  d.  Chem.  Gesellsch.,  34. 

'Walter,  Zeitschr.  f.  physiol.  Chem.,  15;  Levene,  ibid.,  32;  Hammarsten,  not 
published. 


42S  ORGANS  OF  GENERATION. 

to  the  pure  vitellin  isolated  by  Hammarsten  from  perch  eggs.  The  codfish 
ichthulin  yielded  a  pseudonucleic  acid  with  10.34  per  cent  phosphorus,  but  this 
acid  still  gave  the  proteid  reactions. 

The  yolk  also  contains  albumin,  besides  vitellin  and  the  above-mentioned 
globulin. 

The  fat  of  the  yolk  of  the  egg  is,  according  to  Liebermann,  a  mixture 
of  a  solid  and  a  liquid  fat.  The  solid  fat  consists  chiefly  of  tripalmitin  with 
some  stearin.  On  the  saponification  of  the  egg-oil  Liebermann  obtained 
40  per  cent  oleic  acid,  38.04  per  cent  palmitic  acid,  and  15.21  per  cent  stearic 
acid.  The  fat  of  the  yolk  of  the  egg  contains  less  carbon  than  other  fats, 
which  may  depend  upon  the  presence  of  monoglycerides  and  diglycerides,  or 
"upon  a  quantity  of  fatty  acid  deficient  in  carbon  (Liebermann).  In  the 
lecithin,  or  more  correctly  in  the  lecithin  mixture  of  the  yoke,  Cousin  finds 
also  linolic  acid  besides  the  three  ordinary  fatty  acids.  The  composition 
of  yolk  fat  is  dependent  upon  the  food,  as  Henriques  and  Hansen  *  have 
shown  that  the  fat  of  the  food  passes  into  the  egg. 

Lutein.  Yellow  or  orange-red  amorphous  coloring-matters  occur  in  the 
yellow  of  the  egg  and  in  several  other  places  in  the  animal  organism;  for 
instance,  in  the  blood-serum  and  serous  fluids,  fatty  tissues,  milk-fat,  corpora 
lutea,  and  in  the  fat-globules  of  the  retina.  These  coloring-matters,  which 
also  occur  in  the  vegetable  kingdom  (Thudichum),  and  whose  relationship 
to  the  vegetable  pigments,  the  xanthophyll  group,  has  recently  been  shown 
by  Schunck  2,  have  been  called  luteins  or  lipochromes. 

The  luteins,  which  among  themselves  show  somewhat  different  proper- 
ties, are  all  soluble  in  alcohol,  ether,  and  chloroform.  They  differ  from  the 
bile-pigment,  bilirubin,  in  that  they  are  not  separated  from  their  solution 
in  chloroform  by  water  containing  alkali,  and  also  in  that  they  do  not  give 
the  characteristic  play  of  colors  with  nitric  acid  containing  a  little  nitrous 
acid,  but  give  a  transient  blue  color,  and  lastly  they  ordinarily  show  an 
abso  ption-spectrum  of  two  bands,  of  which  one  covers  the  line  F  and  the 
other  lies  between  the  lines  F  and  G.  The  luteins  withstand  the  action 
of  alkalies  so  that  they  are  not  changed  when  we  remove  the  fats  present  by 
means  of  saponification. 

Lutein  has  not  been  prepared  pure.  Maly  3  has  found  two  pigments  free  from 
iron  in  the  eggs  of  a  water-spider  (Maja  sqvinado) — one  a  red  (vitellorubin)  and 
the  other  a  yellow  pigment  (vitdlolutcin) .  Both  of  these  pigments  are  colored 
blue  by  nitric  acid  containing  nitrous  acid  and  beautifully  green  by  concentrated 
sulphuric  acid.  The  absorption-bands,  especially  of  the  vitellolutein,  correspond 
very  nearly  to  those  of  ovolutein. 


1  Cousin,  Compt.  rend.,  137;  Henriques  and  Hansen,  Skand.  Arch.  f.  physiol.,  14. 

2  Thudichum,  Centralbl.  f.  d.  med.  Wissensch.,    1869;    Schunck,  see  Chem.  Cen- 
tralhl.,  1903,  2,  1105. 

8Monatshefte  f.  Chem.,  2. 


THE   WHITE  OF   THE  EGG.  429 

The  mineral  bodies  of  the  yolk  of  the  egg  consist,  according  to  PoLBCK,1 
of  51.2-65.7  parts  soda,  80.5-89.3  potash,  122.1-132.8  lime,  20.7-21.1 
magnesia,  11.90-14.5  iron  oxide,  G38.1-667.0  phosphoric  acid,  and  5.5-14.0 
parts  silicic  acid  in  1000  parts  of  the  ash.  We  find  phosphoric  acid  and 
lime  the  most  abundant,  and  then  potash,  which  is  somewhat  greater  in 
quantity  than  the  soda.  These  results  are  not,  however,  quite  correct;  first, 
because  no  dissolved  phosphate  occurs  in  the  yolk  (Liebermaxx),  and 
secondly,  in  burning,  phosphoric  and  sulphuric  acids  are  produced  and  these 
drive  away  the  chlorine,  which  is  not  accounted  for  in  the  preceding 
analyse-;. 

The  yolk  of  the  hen's  egg  weighs  about  12-18  grams.  The  quantity 
of  water  and  solids  amounts,  according  to  Parkes,2  to  471.9  p.  m.  and 

528.1  p.  m.  respectively.  Among  the  solids  he  found  156.3  p.  m.  proteid, 
3.53  p.  m.  soluble  and  6.12  p.  m.  insoluble  salts.  The  quantity  of  fat, 
according  to  Parkes,  Is  228.4  p.  m.,  the  lecithin,  calculated  from  the  amount 
of  phosphorus  in  the  organic  substance  of  the  alcohol-ether  extract,  was 

107.2  p.  m.  and  the  cholesterin  17.5  p.  m. 

The  white  of  the  egg  is  a  faintly  yellow  albuminous  fluid  enclosed  in  a 
framework  of  thin  membranes;  and  this  fluid  is  in  itself  very  liquid,  but 
seems  viscous  because  of  the  presence  of  these  fine  membranes.  That  sub- 
stance which  forms  the  membranes,  and  of  which  the  chalaza  consists,  seems 
to  be  a  body  nearly  related  to  horn  substances  (Liebermaxn). 

The  white  of  the  egg  has  a  specific  gravity  of  1.045  and  always  has  an 
alkaline  reaction  towards  litmus.  It  contains  850-880  p.  m.  water,  100-130 
p.  m.  proteid  bodies,  and  7  p.  m.  salts.  Among  the  extractive  bodies 
Lbhmann  found  a  fermentable  variety  of  sugar  which  amounted  to  5  p.  m. 
or,  according  to  Meissner,  80  p.  m.  of  the  solids.3  Besides  these  one  finds 
in  the  white  of  the  egg  traces  of  fats,  soaps,  lecithin,  and  cholesterin. 

The  white  of  the  egg  of  the  Insessores  becomes  transparent  on  boiling  and  acts 
in  many  respects  like  alkali  albuminate.  This  albumin  Tarchanoff  *  called 
"tatal'mmin." 

The  protein  substances  of  the  white  of  egg  are  all  glucoproteids,  as  they 
all  yield  glucosamine.  According  to  the  solution  and  precipitation  prop- 
erties they  are  similar  to  the  globulins,  albumins,  or  proteoses.  The  repre- 
sentatives of  the  first  two  groups,  which  until  recently  were  considered 
as  true  proteids,  are  ovoglobulin  and  ovalbumin.  The  proteose-like  body 
is  ovomucoid. 

Ovoglobulin  separates  in  part  on  diluting  the  egg-white  with  water. 
It  is  precipitated  upon  saturation   with   magnesium  sulphate    or  upon 

1  Cited  from  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufi.,  740. 

2  Hoppe-Sevler,  Med.  chem.  Untersuch.,  Heft  2,  209. 
8  Cited  from  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  739. 
*  Pfliiger's  Arch.,  31,  33,  and  39. 


430  ORGANS  OF  GENERATION. 

« 

one  half  saturation  with  ammonium  sulphate  and  coagulates  at  about  75°  C. 
By  repeated  solution  in  water  and  precipitation  with  ammonium  sulphate  a 
part  of  the  globulin  becomes  insoluble  (Langstein).  This  also  occurs  on 
precipitation  by  diluting  with  water  or  by  dialysis  and  it  is  quite  possible 
that  the  globulin  is  a  mixture.  That  portion  which  readily  becomes  in- 
soluble seems  to  be  identical  with  Eichholz's  glucoproteid  or  Osborne  and 
Campbell's  ovomucin.  Langstein  obtained  11  per  cent  of  glucosamine 
from  the  soluble  ovoglobulin.  The  total  quantity  of  globulins,  according 
to  Dillner,  is  about  6.7  per  cent  of  the  total  protein  substances,  and  this 
corresponds  with  the  recent  determinations  of  Osborne  and  Campbell. 
In  regard  to  the  probable  occurrence  of  several  globulins  in  the  white  of 
the  egg  there  are  the  statements  of  Corin  and  Berard  as  well  as  of  Lang- 
stein/ but  they  have  not  led  to  any  positive  conclusions. 

Ovalbumin.  The  so-called  albumin  of  the  egg-white  is  undoubtedly 
a  mixture  of  at  least  two  albumin-like  glucoproteids.  The  views  differ 
considerably  in  regard  to  the  number  of  these  compound  proteids  (Bond- 
zynski  and  Zoja,  Gautier,  Bechamp,  Corin  and  Berard,  Panormoff, 
and  others).  Since  Hofmeister  has  been  able  to  prepare  ovalbumin  in  a 
crystalline  form,  and  since  Hopkins  and  Pinkus  2  have  shown  that  not 
more  than  one  half  of  the  ovalbumin  can  be  obtained  in  such  a  form, 
Osborne  and  Campbell  have  isolated  two  different  ovalbumins  or  chief 
fractions;  the  crystallizable  they  call  ovalbumin  and  the  non-crystallizable, 
conalbumin.  Both  fractions  have  only  a  slight  variation  in  elementary 
composition;  the  conalbumin  coagulates  between  50-60°  C,  nearer  to  60°  C, 
and  the  ovalbumin  at  64°  C.  or  at  a  higher  temperature.  There  are  no  con- 
clusive investigations  as  to  the  point  whether  the  non-crystallizable  con- 
albumin is  a  mixture  or  not,  and  the  question  concerning  the  unity  of  the 
crystallizable  ovalbumin  is  also  disputed.  According  to  Bondzynski 
and  Zoja  crystallizable  ovalbumin  is  a  mixture  of  several  albumins  having 
somewhat  different  coagulation  temperatures,  solubility,  and  specific  rota- 
tion, while  Hofmeister  and  Langstein  on  the  contrary  believe  that  crys- 
tallizable ovalbumin  is  a  unit.  The  statements  as  to  the  specific  rotation 
of  the  different  fractions  unfortunately  differ  and  the  elementary  analyses 
have  also  given  no  positive  results,  as  a  variation  of  1.2-1.7  per  cent  have 
been  observed  in  the  quantity  of  sulphur.  According  to  the  consistent 
analyses  of  Osborne  and  Campbell  and  of  Langstein  the  conalbumin  con- 

1  Langstein,  Hofmeister 's  Beitriige,  1;  Eichholz,  Journ.  of  Physiol.,  23;  Osborne 
and  Campbell,  Connecticut  Agric.  Exp.  Station,  23d  Ann.  Report,  New  Haven,  1900; 
Dillner,  Maly's  Jahresber.,  15;   Corin  and  Berard,  ibid.,  18. 

2  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  11,  16,  and  21;  Gabriel,  ibid.,  15;  Bond- 
zynski and  Zoja,  ibid.,  19;  Gautier,  Bull.  soc.  chim.,  11;  Bechamp,  ibid.,  21;  Corin 
and  Berard,  1.  c. ;  Hopkins  and  Pinkus,  Ber.  d.  d.  chem.  Gesellsch.,  31,  and  Journ.  of 
Physiol.,  23;  Osborne  and  Campbell,  1.  c. ;  Panormoff,  Maly's  Jahresber.,  2?  and  28. 


OVALBUMIX   .LVD  OVOMUCOID.  431 

tains  about  1.7  per  cent  sulphur  and  about  16  percent  nitrogen,  while  the 
ovalbumin  contains  on  an  average  about  15.3  per  cent  nitrogen.  Lang- 
stein  '  obtained  10-11  per  cent  glucosamine  from  ovalbumin  and  about 
0  per  cent  from  conalbumin.  The  ovalbumin  like  the  conalbumin  has 
the  properties  of  the  albumins  in  general,  but  differs  from  seralbumin  in 
the  following:  The  specific  rotation  is  lower.  It  is  made  quickly  insoluble 
by  alcohol  and  is  precipitated  by  a  sufficient  quantity  of  IK'l,  but  dissolves 
in  an  excess  of  acid  with  greater  difficulty  than  the  seralbumin. 

In  preparing  crystalline  ovalbumin  mix,  according  to  Hofmeister,  the 
beaten  white  of  egg  free  from  foam  with  an  equal  volume  of  a  saturated 
ammonium-sulphate  solution,  filter  off  the  globulin,  and  allow  the  filtrate 
to  slowly  evaporate  in  thin  layers  at  the  temperature  of  the  room.  After 
a  time  the  masses  which  separate  out  are  dissolved  in  water,  treated  with 
ammonium-sulphate  solution  until  they  begin  to  get  cloudy,  and  allowed 
to  stand.  After  repeated  recrystallization  the  mass  is  either  treated  with 
alcohol,  which  makes  the  crystals  insoluble,  or  they  are  dissolved  in  water 
ami  purified  by  dialysis.  From  these  solutions  the  proteid  does  not  crys- 
tallize again  on  spontaneous  evaporation.  (See  also  page  430,  foot-note  2, 
for  the  Hopkins  and  Pinkus  method.) 

Conalbumin  can  be  removed  from  the  filtrate,  after  the  complete  crys- 
tallization of  the  ovalbumin,  by  removing  the  sulphate  by  means  of  dialysis 
and  coagulating  by  heat. 

Gautier  2  found  a  fibrinogen-like  substance  in  the  white  of  the  egg,  which 
was  changed  into  a  fibrin-like  body  by  the  action  of  a  ferment. 

Ovomucoid.  This  substance,  first  observed  by  Xeumeister  and  consid- 
ered by  him  as  a  pseudopeptone  and  then  later  studied  by  Salkowski,  is, 
according  to  C.  Tu.  Morxer,3  a  mucoid  with  12.65  per  cent  nitrogen  and 
2.20  per  cent  sulphur.  On  boiling  with  dilute  mineral  acids  it  yields  a 
reducing  substance.  Ovomucoid  exists  in  hens'  eggs  to  the  extent  of  about 
10  per  cent  of  the  total  solids. 

A  solution  of  ovomucoid  is  not  precipitated  by  mineral  acids  nor  by 
organic  acids,  with  the  exception  of  phosphotungstic  acid  and  tannic  acid. 
It  is  not  precipitated  by  metallic  salts,  but  basic  lead  acetate  and  ammonia 
render  it  insolubb.  Ovomucoid  is  thrown  down  by  alcohol,  but  sodium  chlo- 
ride, sodium  sulphate,  and  magnesium  sulphate  give  no  precipitates  either  at 
the  ordinary  temperature  or  when  the  salts  are  added  to  saturation  at  30°  C. 
Its  solutions  are  not  precipitated  by  an  equal  volume  of  a  saturated  solution 
of  ammonium  sulphate,  but  are  precipitated  on  adding  more  salt  thereto. 

1  Zeitschr.  f.  physiol.  Chem.,  31. 

2  Compt.  rend.,  135. 

3  R.  Xeumeister,  Zeitschr.  f.  Biologie,  27:  Salkowski,  Centralbl.  f.  d.  med  Wis- 
sensch.,  1S93,  513  and  70G;  C.  Morner,  Zeitschr.  f.  physiol.  Chem.,  18;  see  also 
Langstein,  Hofmeister's  Beitriige,  3  (literature). 


432  ORGANS  OF  GENERATION. 

The  substance  is  not  precipitated  on  boiling,  but  the  part  which  has  become 
insoluble  in  cold  water  and  then  dried  is  dissolved  by  boiling  water.  Za- 
netti  has  prepared  glucosamine  on  splitting  ovomucoid  with  concentrated 
hydrochloric  acid,  and  Seemann  found  that  the  quantity  of  glucosamine  in 
ovomucoid  was  34.9  per  cent.1 

Ovomucoid  may  be  prepared  by  removing  all  the  proteids  by  boiling 
with  the  addition  of  acetic  acid  and  then  concentrating  the  filtrate  and 
precipitating  with  alcohol.  The  substance  is  purified  by  repeated  solution 
in  water  and  precipitating  with  alcohol. 

The  mineral  bodies  of  the  white  of  the  egg  have  been  analyzed  by  Poleck 
and  Weber.2  They  found  in  1000  parts  of  the  ash:  276.6-284.5  grams 
potash,  235.6-329.3  soda,  17.4-29  lime,  16-31.7  magnesia,  4.4-5.5  iron 
oxide,  238.4-285.6  chlorine,  31.6-48.3  phosphoric  acid  (P205),  13.2-26.3 
sulphuric  acid,  2.8-20.4  silicic  acid,  and  96.7-116  grams  carbon  dioxide. 
Traces  of  fluorine  have  also  been  found  (Nickles  3).  The  ash  of  the  white 
of  the  egg  contains,  as  compared  with  the  yolk,  a  greater  amount  of  chlorine 
and  alkalies  and  a  smaller  amount  of  lime,  phosphoric  acid,  and  iron. 

The  Shell-membrane  and  the  Egg-shell.  The  shell-membrane  consists, 
as  above  stated  (page  57) ,  of  a  keratin  substance.  The  shell  contains  very 
little  organic  substance,  36-65  p.  m.  The  chief  mass,  more  than  900  p.  m., 
consists  of  calcium  carbonate;  besides  this  there  are  very  small  amounts  of 
magnesium  carbonate  and  earthy  phosphates. 

The  diverse  coloring  of  birds '  eggs  is  due  to  several  different  coloring-matters. 
Among  these  we  find  a  red  or  reddish-brown  pigment  called  ' '  oorodein ' '  by  Sorby,4 
which  is  perhaps  identical  with  haematoporphyrin.  The  green  or  blue  coloring- 
matter,  Sorby  's  oocyan,  seems,  according  to  Liebermann5  and  Krukenberg,  8 
to  be  partly  biliverdin  and  partly  a  blue  derivative  of  the  bile-pigments. 

The  eggs  of  birds  have  a  space  at  their  blunt  end  filled  with  gas;  this 
gas  contains  on  an  average  18.0-19.9  per  cent  oxygen  (Hufner  7). 

The  weight  of  a  hen 's  egg  varies  between  40-60  grams  and  may  some- 
times weigh  70  grams.  The  shell  and  shell-membrane  together,  when  care- 
fully cleaned,  but  still  in  the  moist  state,  weigh  5-8  grams.  The  yolk  weighs 
12-18  and  the  white  23-34  grams,  or  about  double.  The  entire  egg  con- 
tains 2.8-7.5,  or  average  4.6  milligrams  of  iron  oxide,  and  the  quantity  of 
iron  can  be  increased  by  food  rich  in  iron  (Hartung  8). 

1  Zanetti,  Chem.  Centralbl.  1898,  1;  Seemann,  cited  from  Langstein,  Ergebnisse 
der  Physiol.,  1,  Abt.  I,  86. 

2  Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  778. 

3  Compt.  rend. ,  43. 

4  Cited  from  Krukenberg,  Verh.  d.  phys.-chem.  Gesellsch.  in  Wurzburg.  17 
6  Ber.  d.  deutsch.  chem.  Gesellsch.,  11. 

8L.  c. 

7Du  Bois-Reymond's  Arch.,  1892. 

8  Zeitschr.  f.  Biologie,  43. 


OVALBUMIN  AND  OVOMUCOID.  433 

The  white  of  t ho  egg  of  cartilaginous  and  bony  fishes  contains  only  truce.  ,,f 
true  albumin,  and  the  cover  of  the  frog's  egg  consists,  according  to  Giacosa,1  of 
mucin.    The  crystalline  formations  (yolk-spherules,  or  dotterpl&ttchen)  which  have 

been  observed  in  the  egg  of  the  tortoise,  frog,  ray,  shark,  and  other  fishes,  and 
Which  are  described  by  VALENCIENNES  and  FSBMT  a  under  the  names  emydin, 
ichthin,  ichthidin,  and  ichthulin,  seem,  as  above  stated  in  connection  with  ichthulin, 
to  consist  chiefly  of  phosphoglucoproteids.  The  egg  of  the  river-crab  and  the 
lobster  contain  the  same  pigment  as  the  shell  of  the  animal.  This  pigment,  called 
cyanocri/stnlUn,  becomes  red  on  boiling  in  water. 

C.  Morneb*  has  isolated  a  substance  which  he  calls  percaglobulin  from  the 
unripe  eggs  of  the  river-perch.  It  is  a  globulin  and  has  a  strong  astringent  taste, 
is  rather  rich  in  sulphur,  1.92  per  cent,  and  is  precipitated  by  0.75  per  cent  HC1. 
Especially  striking  is  its  property  of  precipitating  certain  glucoproteids,  such  as 
ovomucoid  and  ovarial  mucoids,  and  polysaccharides,  such  as  glycogen,  gum  traga- 
canth  or  quince-seed  gum  and  starch-paste,  and  of  being  precipitated  by  them. 

In  fossil  eggs  (of  aptenodytes,  pelecanus,  and  halljsus)  in  old  guano 
deposits,  a  yellowish-white  silky,  laminated  combination  has  been  found  which 
is  called  guanovulit,  (XH4)2S04+2K2S04+3KHS04+4H20,  and  which  is  easily 
soluble  in  water,  but  is  insoluble  in  alcohol  and  ether. 

Those  eggs  which  chvelop  outside  of  the  mother-organism  must  contain 
all  the  elements  necessary  for  the  young  animals.  One  finds,  therefore,  in 
the  yolk  and  white  of  the  egg  an  abundant  quantity  of  proteid  bodies  of 
different  kinds,  and  especially  phosphorized  proteids  in  the  yolk.  Further, 
we  also  find  lecithin  in  the  yolk,  which  seems  habitually  to  occur  in  the 
developing  cell.  The  occurrence  of  glycogen  is  doubtful,  and  the  carbo- 
hydrates are  perhaps  represented  by  a  very  small  amount  of  sugar  and 
glucoproteids.  On  the  contrary,  the  egg  contains  a  large  proportion  of  fat, 
which  doubtless  is  an  important  source  for  the  supply  of  nourishment  and 
in  maintaining  respiration  for  the  embryo.  The  cholasterin  and  the  lutein 
can  hardly  have  a  direct  influence  on  the  development  of  the  embryo. 
The  egg  also  seems  to  contain  the  mineral  bodies  necessary  for  the 
development  of  the  young  animal.  The  lack  of  phosphoric  acid  is  com- 
pensated by  an  abundant  amount  of  phosphorized  organic  substance,  and 
the  nucleoalbumin  containing  iron,  from  which  the  hsematogen  (see  page 
427)  is  formed,  is  doubtless,  as  Bunge  claims,  of  great  importance  in  the 
formation  of  the  haemoglobin  containing  iron.  The  silicic  acid  necessary 
for  the  development  of  the  feathers  is  also  found  in  the  egg. 

During  the  period  of  incubation  the  egg  loses  weight,  chiefly  due  to  loss 
of  water.  The  quantity  of  solids,  especially  the  fat  and  the  proteids, 
diminishes  and  the  egg  gives  off  not  only  carbon  dioxide,  but  also,  as 
Liebekmaxx  ''"has  shown,  nitrogen  or  a  nitrogenous  substance.  The  loss 
is  compensated  by  the  absorption  of  oxygen,  and  it  is  found  that  during 
neubation  a  respirator}-  exchange  of  gas  takes  place. 

1  Zeitschr.  f.  physiol.  Chem.,  7. 

'Cited  from  Hoppe-Seyler's  Physiol.  Chem.,  77. 

8  Zeitschr.  f.  physiol.  Chem.,  40. 

♦Pfluger's  Arch",  43. 


434  ORGANS  OF  GENERATION. 

As  Bohr  and  Hasselbach  have  shown  by  exact  investigations,  the 
elimination  of  carbon  dioxide  is  very  small  the  first  days  of  incubation; 
on  the  fourth  day  the  carbon  dioxide  production  gradually  increases  and 
after  the  ninth  day  it  augments  in  the  same  proportion  as  the  weight  of 
the  foetus.  Calculated  upon  1  kilogram  weight  for  one  hour  it  is,  from  the 
ninth  clay  on,  about  the  same  as  in  the  full  grown  hen.  Hasselbach  1 
has  also  shown  that  the  fertilized  hen's  egg  not  only  gives  off  nitrogen 
the  first  five  or  six  hours  of  incubation,  but  also  some  oxygen,  and  that  we 
are  here  dealing  with  an  oxygen  production  which  runs  parallel  with  the 
cell-division.  It  is  not  known  whether  this  oxygen  formation  connected 
with  the  life  of  the  cell  is  a  fermentative  or  a  so-called  vital  process. 

While  the  quantity  of  dry  substance  in  the  egg  during  this  period  always 
decreases,  the  quantity  of  mineral  bodies,  proteid,  and  fat  always  increases 
in  the  embryo.  The  increase  in  the  amount  of  fat  in  the  embryo  depends, 
according  to  Liebermann,  in  great  part  upon  a  taking  up  of  the  nutritive 
yolk  in  the  abdominal  cavity.  The  weight  of  the  shell  and  the  quantity  of 
lime-salts  contained  therein  remains  unchanged  during  incubation.  The 
yolk  and  white  together  contain  the  necessary  quantity  of  lime  for  devel- 
opment. 

The  most  complete  and  careful  chemical  investigation  on  the  develop- 
ment of  the  embryo  of  the  hen  has  been  made  by  Liebermann.  From  his 
researches  we  may  quote  the  following :  In  the  earlier  stages  of  the  develop- 
ment, tissues  very  rich  in  water  are  formed,  but  upon  the  continuation  of  the 
development  the  quantity  of  water  decreases.  The  absolute  quantity  of 
the  bodies  soluble  in  water  increases  with  the  development,  while  their  rela- 
tive quantity,  as  compared  with  the  other  solids,  continually  decreases. 
The  quantity  of  the  bodies  soluble  in  alcohol  quickly  increases.  A  specially 
important  increase  is  noticed  in  the  fat,  whose  quantity  is  not  very  great 
even  on  the  fourteenth  day,  but  after  that  it  becomes  considerable.  The 
quantity  of  proteid  bodies  and  albuminoids  soluble  in  water  grows  contin- 
ually and  regularly  in  such  a  way  that  their  absolute  quantity  increases, 
while  their  relative  quantity  remains  nearly  unchanged.  Liebermann 
found  no  gelatine  in  the  embryo  of  the  hen.  The  embryo  does  not  contain 
any  gelatine-forming  substance  until  the  tenth  day,  and  from  the  fourteenth 
day  on  it  contains  a  body  which  when  boiled  with  water  gives  a  substance 
similar  to  chondrin.  A  body  similar  to  mucin  occurs  in  the  embryo  when 
about  six  days  old,  but  then  disappears.  The  quantity  of  haemoglobin 
shows  a  continual  increase  compared  with  the  weight  of  the  body.  Lieber- 
mann found  that  the  relationship  of  the  haemoglobin  to  the  body  weight  was- 
1 :728  on  the  eleventh  day  and  1 :421  on  the  twenty-first  day. 


1  Bohr   and   Hasselbach,  Maly's   Jahresber.,   29;    Hasselbach,    Skand.    Arch.    f. 
Physiol.,  13. 


PLACENTA.     AMNIOTIC  FLUID.  435 

By  means  of  Berthelot's  thermometric  methods  Tangl  has  deter- 
mined the  chemical  energy  present  at  the  beginning  and  end  of  the  develop- 
ment of  the  embryo  of  the  sparrow's  and  hen's  eggs.  The  difference  was 
considered  as  workof  development.  He  found  thai  the  chemical  energy 
necessary  fo]  the  development  of  1  gram  of  ripe  or  nearly  ripe  lien's  embryo 
(Plymouth  egg)  was  equal  to  658  Calories.  This  energy  originated  chiefly 
from  the  fat.  <  >f  the  total  chemical  energy  utilized,  two  thirds  was  used  tor 
the  construction  of  the  embryo  and  one  third  transformed  into  other  forms 
of  energy  as  work  of  development.  Still  more  recent  researches  of  Bohr  and 
Hasselbach  'show  that  none  of  the  transformed  chemical  energy  is  used  in 
the  construction  of  the  embryo,  as  it  nearly  entirely  leaves  the  egg  as  heat. 

The  tissue  of  the  placenta  has  not  thus  far  boon  the  subject  of  detailed  chemical 
investigation.  In  the  edges  of  the  placenta  of  bitches  and  of  cats  a  crystallizable 
orange-colored  pigmenl  (biluribin?)  has  been  found,  and  also  a  green  amorphous 
pigment,  Meckel's  hoematochlorin,  which  is  considered  as  biliverdin  by  Eui.- 
Peeyer'  questions  the  identity  of  these  pigments  with  biliverdin. 

From  the  cotyledons  of  the  placenta  in  ruminants  a  white  or  faintly  rose-colored 
creamy  Quid,  the  uterine  milk,  can  be  obtained  by  pressure.  It  is  alkaline  in 
reaction,  but  becomes  acid  quickly.  Its  specific  gravity  is  1.033-1.040.  It  con- 
tains us  form-elements  fat-globules,  small  granules,  and  epithelium-cells.  There 
has  been  found  81.2-120.9  p.  m.  solids,  01.2-105.6  p.  m.  proteid,  about  10  p.  m. 
fat,  and  3.7-8.2  p.  m.  ash  in  the  uterine  milk. 

The  fluid  occurring  in  the  so-called  grape-mole  (Mola  racemosa)  has  a  low 
specific  gravity,  1.009-1.012,  and  contains  19.4-26.3  p.  m.  solids  with  9-10  p.  m. 
protein  bodies  and  6-7  p.  m.  ash. 

The  amniotic  fluid  in  women  is  thin,  whitish,  or  pale  yellow;  sometimes 
it  is  somewhat  yellowish-brown  and  cloudy.  White  flakes  separate.  The 
form-elements  are  mucus-corpuscles,  epithelium-cells,  fat-drops,  and  lanugo 

hair.     The  odor  is  stale,  the  reaction  neutral  or  faintly  alkaline.     The 
specific  gravity  is  1.002-1.028. 

The  amniotic  fluid  contains  the  constituents  of  ordinary  transudates. 
The  amount  of  solids  at  birth  is  hardly  20  p.  m.  In  the  earlier  stages  of 
pregnancy  the  fluid  contains  more  solids,  especially  proteids.  Among  the 
prote':d  bodies  Weyl  found  one  substance  similar  to  vitellin,  and  with  great 
probability  also  seralbumin,  besides  small  quantities  o  mucin.  Enzymes 
of  various  kinds  (pepsin,  diastase,  thrombin,  lipase)  occur  according  to 
Bondi.  Sugar  is  regularly  found  in  the  amniotic  fluid  of  cows,  but  not  in 
human  beings.  On  the  contrary,  the  human  amniotic  fluid  contains  some 
urea  and  allantoin.  The  quantity  of  these  may  be  increased  in  hydramnion 
(Prochownick,  Harnack),  which  depends  on  an  increased  secretion  by 
the  kidneys  and  skin  of  th  •  fostus.  Creatine  and  lactates  are  doubtful  con- 
stituent df  the  amniotic  fluid..     The  quantity  of  urea  in  the  amniotic  fluid 

1  Tangl.  Pfluger's  Arch.,  93;   Bohr  and  Hasselbach,  Skand.  Arch.  f.  Physiol.,  14. 

,Maly's  Jahrosbcr.,  2,  287. 

3  Die  Rlutkristalle.  Jena,  1871,  1S9. 


436  ORGANS  OF  GENERATION. 

is,  according  to  Prochownick,  0.16  p.  m.  In  the  fluid  in  hydramnion 
Prochownick  and  Harnack  found  respectively  0.34  and  0.48  p.  m.  urea. 
The  chief  mass  of  the  solids  consists  of  salts.  The  quantity  of  chlorides 
(NaCl)  is  5.7-6.6  p.  m.  The  molecular  concentration  of  the  amniotic  fluid 
is  somewhat  lower  than  that  of  the  blood,  which  is  no  doubt  due  to  a 
dilution  by  the  foetal  urine  (Zangemeister  and  Meissl  1). 

1  Weyl,  Du  Bois-Reymond  's  and  Reichert's  Arch. ,  1876 ;  Bondi,  Centralbl.  f .Gynakol. , 
1903;  Prochownick,  Arch.  f.  Gynak.,  11,  also  Maly's  Jahresber.,  7,  155;  Harnack, 
Berlin  klin.  Wochenschr. ,  1888,  No.  41;  Zangemeister  and  Meissl,  Munch,  med.  Woch- 
enschr.,  1903. 


CHAPTER  XIV. 
MILK. 

The  chemical  constituents  of  the  mammary  glands  have  been  little 
studied.  The  cells  are  rich  in  proteid  and  nucleoproteids,  Among  the  latter 
we  have  one  that  yields  pentose  and  guanine,  but  no  other  purin  base  on 
boiling  with  dilute  mineral  acids.  This  compound  proteid,  investigated  by 
(  )di:xius,  contains  as  an  average  the  following:  17.28  per  cent  X,  0.89  per 
cent  S,  and  0.277  per  cent  P.  One  cannot  state  what  relation  this  body 
bears  to  that  constituent  of  the  gland  found  by  Bert,  which  on  boiling  with 
dilute  mineral  acids  yielded  a  reducing  substance.  Such  a  substance, 
which  acts  perhaps  as  a  step  towards  the  formation  of  lactose,  has  also 
been  observed  by  THiERFELDERj^^Faf  seems  to  be  a  never-failing  con- 
stituent of  the  secreting  gland,  and  this  fat  may  be  observed  in  the 
protoplasm  as  large  or  small  globules  similar  to  milk-globules.  The  ex- 
tractive bodies  of  the  mammary  glands  have  been  little  investigated,  but 
among  them  are  found  considerable  amounts  of  purin  basely 

As  human  milk  and  the  milk  of  animals  are  essentially  of  the  same 
constitution,  it  seems  best  to  speak  first  of  the  one  most  thoroughly  inves- 
tigated, namely,  cow's  milk,  and  then  of  the  essential  properties  of  the 
remaining  important  kinds  of  milk.2 

Cow's  Milk. 

y  Cow's  milk,  like  every  other  kind,  forms  an  emulsion  which  consists  of 
very  finely  divided  fat  suspended  in  a  solution  consisting  chiefly  of  proteid 
bodies,  milk-sugar,  and  salts.  Milk  Is  non-transparent,  white,  whitish 
yellow,  or  in  thin  layers  somewhat  bluish-white,  of  a  faint,  insipid  odor  and 
mild,  faintly  sweetish  taste.  The  specific  gravity  is  1.028  to  1.0345  at 
15°  C.^  The  freezing-point  is  0.54-0.59°  C,  average  0.563°  C„  and  the  mo- 
lecular concentration  0.298. 

The  reaction  of  perfectly  fresh  milk  is  generally  amphoteric  towards 
litmus.     The  extent  of  the  acid  and  alkaline  part  of  this  amphoteric  reac- 


^denius,  Maly's  Jahresber.,  30;    Bert,  Compt.  rend.,  98;    Thierfelder,  Pfluger's 
Arch.,  32,  and  Maly's  Jahresber.,  13. 

2  A  very  complete  reference  to  the  literature  on  milk  may  be  found  in  Raudnitz'a 
"Die  Bestandteile  der  Milch,"  in  Ergebnisse  der  Physiol.,  2,  Abt.  I. 

437 


438  MILK 

tion  has  been  determined  by  different  investigators,  especially  Thorner, 
Sebelien,  and  Courant.1     The  results  differ  somewhat  with  the  indicators 
used,  and  moreover  the  milk  from  different  animals,  as  well  as  that  from 
the  same  animal  at  different  times  during  the  lactation  period,  varies 
somewhat.     Courant  has  determined  the  alkaline  part  by  N/10  sulphuric 
acid,  using  blue  lacmoid  as  indicator,  and  the  acid  part  by  N/10  caustic 
soda,  using  phenolphthalein  as  indicator.     He  found,  as  an  average  for  the 
first  and  last  portions  of  the  milking  of  twenty  cows,  that  100  c.  c.  milk 
had   the   same   alkaline   reaction   toward   blue  lacmoid  as  41  c.  c.  N/10 
caustic    soda,    and   the   same    acid   reaction   toward    phenolphthalein   as 
19.5  c.  c.  N/10  sulphuric  acid. 
X^Muk  gradually  changes  when   exposed  to   the  air,   and  its   reaction  I 
becomes  more  and  more  acid.     This  depends  on  a  gradual  transformation/ 
of  the  milk-sugar  into  lactic  acid,  caused  by  micro-organisms.  ^/ 

Entirely  fresh  amphoteric  milk  does  not  coagulate  on  boiling,  but  forms 
a  skin  consisting  of  coagulated  casein  and  lime-salts,  which  rapidly  re- 
forms after  being  removed.  Even  after  passing  a  current  of  carbon  dioxide 
through  the  fresh  milk  it  does  not  coagulate  on  boiling.  In  proportion 
as  the  formation  of  lactic  acid  advances  this  behavior  changes,  and  soon  a 
stage  is  reached  when  the  milk,  which  has  previously  had  carbon  dioxide 
passed  through  it,  coagulates  on  boiling.  At  a  second  stage  it  coagulates 
alone  on  heating ;  then  it  coagulates  by  passing  carbon  dioxide  alone  with- 
out boiling;  and  lastly,  when  the  formation  of  lactic  acid  is  sufficient,  it 
coagulates  spontaneously  at  the  ordinary  temperature,  forming  a  solid 
mass.  It  may  also  happen,  especially  in  the  warmth,  that  the  casein- 
clot  contracts  and  a  yellowish  or  yellowish-green  acid  liquid  (acid  whey) 
separates 


Milk  may  undergo  various  fermentations.  Lactic-acid  fermentation,  brought 
about  by  Huppe's  lactic-acid  bacillus,  and  also  other  varieties  take  first  place. 
In  the  spontaneous  souring  of  milk  we  generally  consider  the  formation  of  lactic 
acid  as  the  most  essential  product,  but  a  formation  of  succinic  acid  may  also  take 
place,  and  in  certain  bacterial  decompositions  of  milk  succinic  acid  and  no  lactic 
acid  is  formed.  The  materials  from  which  these  two  acids  are  formed  are  lactose 
and  laftophosphocarnic  acid.  Besides  lactic  and  succinic  acids,  volatile  fatty 
acids,  such  as  acetic  acid,  butyric  acid,  and  others,  may  be  formed  in  the  bacterial 
decomposition  of  milk. 

Milk  sometimes  undergoes  a  peculiar  kind  of  coagulation,  being  converted 
into  a  thick,  ropy,  slimy  mass  (thick  milk).  This  conversion  depends  upon  a 
peculiar  change  in  which  the  milk-sugar  is  made  to  undergo  a  slimy  transforma- 
tion.    This  transformation  is  caused  by  special  micro-organisms. 

S  If  the  milk  is  sterilized  by  heating  and  contact  with  micro-organisms 

prevented,  the  formation  of  lactic  acid  may  be  entirely  stopped.     The 

production  of  acid  may  also  be  prevented,  at  least  for  some  time,  by  many 

antiseptics,  such  as  salicylic  acid,  thymol,  boric  acid,  and  other  bodies.    / 

1  Thorner,  Maly's  Jahresber.,  22;   Sebelien,  ibid.;   Courant,  Pfliiger's  Arch.,  50. 


MILK-GLOBULES.  439 

If  freshly  drawn  amphoteric  milk  is  treated  with  rennet,  it  coagulates 
quickly,  especially  at  the  temperature  of  the  body,  to  a  solid  mass  (curd) 
from  which  a  yellowish  fluid  (sweet  whey)  is  gradually  pressed  out.  This 
coagulation  occurs  without  any  change  in  the  reaction  of  the  milk,  and 
therefore  it  is  distinct  from  the  acid  coagulation. /* 

In  cow's  milk  we  find  as  form-elements  a  few  colostrum  corpuscles 
(see  Colostrum)  and  a  few  pale  nucleated  cells.  The  number  of  these 
form-elements  is  very  small  compared  with  the  immense  amount  of  the 
most  essential  form-constituents,  the  milk-globules. 

The  Milk-globules.  These  consist  of  extremely  small  drops  of  fat  whose 
number  is,  according  to  Woll,1  1.06-5.75  millions  in  1  c.  mm.,  and  whose 
diameter  is  0.0024-0.0046  mm.  and  0.0037  mm.  as  an  average  for  different 
kinds  of  animals.  It  is  unquestionable  that  the  milk-globules  contain  fat, 
and  we  consider  it  as  positive  that  all  the  milk-fat  exists  in  them.  Another 
disputed  question  is  whether  the  milk-globules  consist  entirely  of  fat  or 
whether  they  also  contain  proteid. 

According  to  the  observations  of  AsCHEHSON,-3  drops  of  fat,  when 
dropped  in  an  alkaline  proteid  solution,  are  covered  with  a  fine  albuminous 
coat,  a  so-called  haptog 'en-membrane.  As  milk  on  shaking  with  ether  does 
not  give  up  its  fat,  or  only  very  slowly  in  the  presence  of  a  great  excess  of 
ether,  and  as  this  takes  place  very  readily  after  the  addition  of  acids  or 
alkalies,  which  dissolve  proteids,  it  was  formerly  thought  that  the  fat- 
globules  of  the  milk  were  enveloped  in  a  proteid  coat.  A  true  membrane 
has  not  been  detected;  and  since,  when  no  means  of  dissolving  the  proteid 
is  resorted  to — for  example,  when  the  milk  is  precipitated  by  carbon  dioxide 
after  the  addition  of  very  little  acetic  acid,  or  when  it  is  coagulated  by 
rennet — the  fat  can  be  very  easily  extracted  by  ether,  the  theory  of  a 
special  albuminous  membrane  for  the  fat-globule  has  been  generally  aban- 
doned. The  observations  of  Quincke  3  on  the  behavior  of  the  fat-globules 
I  in  an  emulsion  prepared  with  gum  have  led,  at  the  present  time,  to  the 
conclusion  that  each  fat-globule  in  the  milk  is  surrounded  by  a  stratum  of 
casein  solution  held  by  molecular  attraction,  and  this  prevents  the  globules 
from  uniting  with  each  other.  Everything  that  changes  the  physical 
condition  of  the  casein  in  the  milk  or  precipitates  it  must  necessarily  help 
the  solution  of  the  fat  in  ether,  and  it  is  in  this  way  that  the  alkalies, 
acids,  and  rennet  act^^ 

Storch  has  shown,  in  opposition  to  these  views,  that  the  milk-globules 
are  surrounded  by  a  membrane  of  a  special  slimy  substance.  This  substance 
is  very  insoluble,  contains  14.2-14.79  per  cent  nitrogen,  and  yields  a  sugar 

1  On  the  Conditions  Influencing  the  Number  and  Size  of  Fat-globules  in  Cow's  Milk, 
Wisconsin  Expt.  Station,  6,  1892. 
'Arch.  f.  Anat.  u.  Physiol.,  1840. 
•Pfluger's  Arch.,19. 


440  MILK. 

or  at  least  a  reducing  substance,  on  boiling  with  hydrochloric  acid.  It  is 
neither  casein  nor  lactalbumin,  but  seems  to  all  appearances  to  be  identical 
with  the  so-called  "stroma  substance"  detected  by  Radenhausen  and 
Danilewsky.  Storch  was  able  to  show  that  this  substance  enveloped 
the  fat-globules  like  a  membrane  by  staining  the  same  with  certain  dyes.1 
v^The  milk-fat  which  is  obtained  under  the  name  of  butter  consists 
chiefly  of  olein  and  palmitin.  Besides  these  it  contains,  as  triglycerides, 
myristic  acid,  stearic  acid,  small  amounts  of  lauric  acid,  arachidic  acid,  and 
dioxystearic  acid,  besides  butyric  acid  and  caproic  acid,  traces  of  caprylic 
acid  and  capric  acid;  also  the  presence  of  mixed  glycerides  (see  Chapter  IV) 
is  not  improbable.  Milk-fat  also  contains  a  small  quantity  of  lecithin  and 
cholesterin,  and  a  yellow  coloring-matter.  The  quantity  of  volatile  fatty 
acids  in  butter  is,  according  to  Duclaux,  on  an  average  about  70  p.  m., 
of  which  37-51  p.  m.  is  butyric  acid  and  20-33  p.  m.  is  caproic  acid.  The 
non-volatile  fat  consists  of  -rV"ro  olein,  and  the  remainder  is  chiefly  palmitin. 
The  composition  of  butter  is  not  constant,  but  varies  considerably  under 
different  circumstances.2 
yr  The  milk-plasma,  or  that  fluid  in  which  the  fat-globules  are  suspended, 
contains  several  different  proteids ;  the  statements  as  to  number  and  nature 
of  which  are  somewhat  at  variance.  The  three  following,  casein,  lactalbumin, 
and  lactglobulin,  have  been  closest  studied  and  are  well  characterized.  The 
milk-plasma  also  contains  two  carbohydrates,  of  which  the  one,  lactose,  is 
of  great  importance.  The  milk-plasma  also  contains  extractive  bodies, 
traces  of  urea,  creatine,  creatinine,  hypoxanthine  (?),  lecithin,  cholesterin f 
citric  acid  (Soxhlet  and  Henkel3),  and  lastly  also  mineral  bodies  and/ 
gases. 

Casein.    This  protein  substance,  which  thus  far  has  been  detected  posi- 

(tively  only  in  milk,  belongs  to  the  nucleoalbumins,  and  differs  from  the  | 
albuminates  chiefly  by  its  content  of  phosphorus  and  by  its  behavior  with  j 
the  rennet  enzymey^Casein  from  cow's  milk  has  the  following  composition: 
C  53.0,  H  7.0,  N  15.7,  S  0.8,  P  0.85,  and  O  22.65  per  cent.  Its  specific 
rotation  is,  according  to  Hoppe-Seyler,4  somewhat  variable;  in  neutral 
solution  it  is  (a)D  =  —80°.  The  question  whether  the  casein  from  different 
kinds  of  milk  is  identical  or  whether  there  are  several  different  caseins 
is  still  disputed. 

Casein  when  dry  appears  like  a  fine  white  powder,  which  has  no  meas- 

1  V.  Storch,  see  Maly's  Jahresber.,  27;  Radenhausen  and  Danilewsky,  Forschungen 
auf  dem  Gebiete  der  Viehhaltung  (Bremen,  1880),  Heft  9. 

2  Duclaux,  Compt.  rend. ,  104.  Various  statements  as  to  the  composition  of  milk- 
fat  can  be  found  in  Koefoed,  Bull.  d.  l'Acad.  Danoise,  1891,  and  Wanklyn,  Chemical 
News,  63;  Browne,  Chera.  Centralbl.,  1899,  2,  833. 

3  Cited  from  F.  Soldner,  Die  Salze  der  Milch,  etc.  Landwirthsch.  Versuchsstation, 
35.     Separatabzug,  18. 

4  Handb.  d.  physiol   u.  pathol.  chem.  Analyse,  G.  Aufl  ,  259. 


CASEIN.  HI 

urable  solubility  in  purr  water  (Laqteur  and  Sackt-k).  Casein  is  only  very 
slightly  soluble  in  the  ordinary  neutral-sail  solutions.  According  to  Annus 
it  dissolves  rather  easily  in  a  1  per  ccnl  solution  of  sodium  fluoride,  ammo- 
nium, or  potassium  oxalate.  It  Is  at  least  a  tetrabasic  acid,  whose  equivalent 
weight  is  LI 35  according  to  Laqueub  and  Sackub  l  and  whose  molecular 
weight  is  four  or  six  times  this.  The  salts  are  split  hydrolytically.  It 
dissolves  readily  in  water  with  the  aid  of  an  alkali  or  alkaline  earths,  also 
calcium  carbonate,  from  which  it  expels  carbon  dioxide/"  If  casein  is  dis-^ 
solved  in  lime-water  and  this  solution  carefully  treated  with  very  dilute 
phosphoric  acid  until  it  is  neutral  in  reaction,  the  casein  appears  to  remain 
in  solution,  but  is  probably  only  swollen  as  in  milk,  and  the  liquid  contains 
at  the  same  time  a  large  quantity  of  calcium  phosphate  without  any  pre- 
cipitate or  any  suspended  particles  being  visible.  The  casein  solutions 
containing  lime  are  opalescent  and  have  on  warming  the  appearance  of 
milk  deficient  in  fat  (which  is  also  true  for  the  salts  of  casein  with  the  alka- 
line earths).  Therefore  it  is  not  impossible  that  the  white  color  of  the  milk^/ 
is  due  partly  to  the  casein  and  calcium  jphosphatey/ISoLDNER  has  pre- 
pared two  calcium  combinations  of  casein  with  1.55  and  2.36  per  cent  CaO, 
and  these  combinations  are  designated  di-  and  tricalcium  casein  by  Cou- 
raxt.2 

Casein  solutions  do  not  coagulate  on  boiling,  but  are  covered,  like  milk, 
with  a  skin.  They  are  precipitated  by  very  little  acid,  but  the  presence  of 
neutral  salts  retards  the  precipitation.  A  casein  solution  containing  salt  or 
ordinary  milk  requires,  therefore,  more  acid  for  precipitation  than  a  salt- 
free  solution  of  casein  of  the  same  concentration.  The  precipitated  casein 
dissolves  very  easily  again  in  a  small  excess  of  hydrochloric  acid,  but  less 
easily  in  an  excess  of  acetic  acid.  These  acid  solutions  are  precipitated  by 
mineral  acids  in  excess^Casein  Is  precipitated  from  neutral  solutions  or  I 

(from  milk  by  common  salt  or  magnesium  sulphate  in  substance  without' 
changing  its  properties/  Metallic  salts,  such  as  alum,  zinc  sulphate,  and 
copper  sulphate,  completely  precipitate  the  casein  from  neutral  solutions. 

On  drying  at  100°  C,  casein,  according  to  Laqueur  and  Sackur,  decom- 
poses and  splits  into  two  bodies.  One  of  these,  called  cascul.  is  insoluble 
in  dilute  alkalies,  while  the  other,  the  isocasein,  Is  soluble  therein.  The  is  - 
casein  is  a  stronger  acid,  and  has  other  precipitation  limits  and  a  somewhat 
L >wcr  equivalent  weight  than  the  casein. 

The  property  which  is  the  mos1  characteristic  of  casein  is  that  it  coagu-| 
Lates  with  rennet  in  the  presence  of  a  sufficiently  great  amount  of  Lime  salts.  | 

1  Laqueur  and  Sackur,  Hofmeister's  Beitriige,  3;  M.  Arthus,  Theses  presentees 
a  la  faculte  dea  sciences  de  Paris,  1S93. 

2  Soldner,  Die  Salze  der  Milch,  etc.;  Courant,  1.  c.  In  regard  to  the  salts  of  casein 
see  the  investigations  of  Soldner,  Mary's  Jahresber.,  85,  and  J.  Rdhmann,  Berlin  klin. 
"Wocbenschr.,  1895.     See  also  Raudnitz,  Ergebnisse  der  Physiol.,  2,  Abt.  I. 


442  MILK. 

In  solutions  free  from  lime  salts  the  casein  does  not  coagulate  with  rennet,  ti 
but  it  is  changed  so  that  the  solution  (even  if  the  enzyme  is  destroyed  by 
heating)  yields  a  coagulated  mass,  having  the  properties  of  a  curd,  if  lime 
salts  are  added.  The  rennet  enzyme,  rennin,  has  therefore  an  action  on 
casein  even  in  the  absence  of  lime  salts,  and  these  last  are  only  necessary 
for  the  coagulation  or  the  separation  ofthecurd/' This  fact,  which  was 
first  proved  by  Hammarsten,  was  later  confirmed  by  Arthus  and  Pages 
and  recently  closely  studied  by  Fuld.1 

The  curd  formed  on  the  coagulation  of  milk  contains  large  quantities  of 
calcium  phosphate.  According  to  Soxhlet  and  Soldner,  the  soluble 
lime  salts  are  of  essential  importance  only  in  coagulation,  while  the  calcium 
phosphate  is  without  importance.  According  to  Courant  the  calcium 
casein  on  coagulation  may  carry  down  with  it,  if  the  solution  contains 
dicalcium  phosphate,  a  part  of  this  as  tricalcium  phosphate,  leaving  mono- 
calcium  phosphate  in  the  solution.  We  are  not  quite  clear  as  to  the  impor- 
tance of  the  lime  salts  for  the  rennin  coagulation  and  the  views  are  still 
somewhat  variable  on  this  question.  The  same  is  true  for  the  chemical 
processes  going  on  in  rennin  coagulation.  If  one  makes  use  of  a  pure  solu- 
tion of  casein  and  as  pure  rennin  as  possible  after  coagulation,  it  is  always 
found  that  the  filtrate  contains  very  small  amounts  of  a  proteid,  the  whey- 
proteid,  which  has  other  properties  and  a  lower  content  of  nitrogen  (13.2  per 
cent  N,  Koster  2),  than  the  casein.  The  chief  portion  of  the  casein,  some- 
times given  as  more  than  90  per  cent,  separates  on  coagulation  as  a  body, 
the  paracasein  (or  curd),  which  is  closely  related  to  casein.  The  question 
whether  a  cleavage  of  the  casein  takes  place  here  is  still  unsettled.  The 
paracasein  3  is  not  further  changed  by  the  rennet  enzyme,  and  it  has  not 
the  property,  to  the  same  extent,  of  holding  calcium  phosphate  in  solution 
as  casein  has.4 

/"""  In  the  digestion  of  casein  with  pepsin-hydrochloric  acid  primarily  a  1 
phosphorized  proteose  is  formed  from  which  then  the  pseudonuclein  is  | 
split  off  (SALKOWSKi^y  The  quantity  thus  split  off  is  very  variable,  as 

1  See  Maly's  Jahresber.,  2  and  4;  also  Hammarsten,  Zur  Kenntniss  des  Kaselns  und 
der  "Wirkung  des  Labfermentes.  Nova  Acta  Reg.  Soc.  Scient.  Upsala,  1877.  Fest- 
schrift; Zeitsehr.  f.  physiol.  Chem.,  22;  Arthus  et  Pages,  Arch,  de  Physiol.  (5),  2,  and 
MY-m.  Soc.  bioL,  43;  Fuld,  Hofmeister's  Beitriige,  2,  and  Ergebnisse  der  Physiol.,  1, 
Abt.  I,  where  a  good  review  of  the  literature  may  be  found. 

2  See  Maly  's  Jahresber. ,  11. 

3  It  has  been  recently  proposed  to  designate  the  ordinary  casein  as  caseinogen  and 
the  curd  as  casein.  Although  such  a  proposition  is  theoretically  correct,  it  leads  in 
practice  to  confusion.  On  this  account  the  author  calls  the  curd  paracasein,  according 
to  Schulze  and  Rose  (Landwirthsch.  Versuchsstat. ,  31). 

*  In  regard  to  recent  work  on  the  coagulation  of  milk,  we  must  mention  Hillmann, 
Milchzeitung,  25;  Benjamin,  Virchow's  Arch  ,  145;  and  Lorcher,  Pfliiger's  Arch.,  (50; 
Fuld,  1.  c. 


DIGESTION  OF  CASEIN.  443 

shown  by  the  researches  of  SALKOWBKl,  IIahv,  MoRACZEWSKJ  ami  Si:iu;- 
LTEN.1  The  amount  of  phosphorus  in  the  pseudonucleins  obtained  also 
varies  considerably.  According  to  SALKOWSK]  the  quantity  of  the  pseu- 
donuclein  split  off  is  dependent  upon  the  relationship  between  the  casein 
and  digestion  fluid,  e.g.,  the  quantity  of  the  pseudonucleins  diminishes  as 
the  pepsin-hydrochloric  acid  increases.  In  the  presence  of  500  grams  of 
pepsin-hydrochloric  acid  to  1  gram  of  casein  Salkowski  digested  the  latter 
completely  without  obtaining  any  pseudonuclein. 

In  peptic  as  well  as  tryptic  digestion  a  part  of  the  organic  phos- 
phorus is  split  off  as  orthophosphoric  acid,  the  quantity  increasing  as 
the  digestion  progresses.  Another  part  of  the  phosphorus  is  retained  in 
organic  combination,  in  the  proteoses  as  well  as  in  the  true  peptone  (Sal- 
kowski,  Biffi,  Alexander  2). 

From  the  products  of  peptic  digestion  of  casein,  after  the  separation  of 
the  pseudonuclein,  Salkowski  3  has  isolated  an  acid  rich  in  phosphorus. 
He  calls  this  paranucleic  acid.  It  is  soluble  in  water,  insoluble  in  alcohol, 
laevorotatorv,  and  has  the  following  composition:  C  42.51-42.96,  H  6.97-7.09, 
N  13.25-13.55,  and  P  4.05-4.31  per  cent.  The  acid  differs  from  the  nucleic 
acids  in  that  it  gives  the  biuret  test  and  a  faint  xanthoproteic  reaction. 
Presupposing  its  purity  it  is  not  an  acid  comparable  to  the  nucleic  acids. 

Casein  may  be  prepared  in  the  following  way:  The  milk  is  diluted  with 
4  vols,  of  water  and  the  mixture  treated  with  acetic  acid  to  0.75  to  1p.m. 
( lasein  thus  obtained  is  purified  by  repeatedly  dissolving  in  water  with  the  aid 
of  the  smallest  quantity  of  alkali  possible,  by  filtering  and  reprecipitating 
with  acetic  acid  and  thoroughly  washing  with  water.  Most  of  the  milk-fat 
is  retained  by  the  filter  on  the  first  filtration,  and  the  casein  contaminated 
with  traces  of  fat  is  purified  by  treating  with  alcohol  and  ether.  y* 

-T  Lactoglobulin  was  obtained  by  Sebeliex  from  cow's  milk  by  saturating 
it  with  NaCl  in  substance  (which  precipitated  the  casein)  and  saturating 

I  the  filtrate  with  magnesium  sulphate/  As  far  as  it  has  been  investigated  it 
had  the  properties  of  serglobulin;  the  globulin  isolated  by  Tiemaxx  *  from 
colostrum  had  nevertheless  a  markedly  low  content  of  carbon,  namely, 
49.83  per  cent. 

Lactalbumin  was  first  prepared  in  a  pure  state  from  milk  by  Sebeliex. 
Its  composition  is,  according  to  him,  C  52.19,  H  7.18,   N  15.77,  S  1.73, 


'Salkowski,  Zeitschr.  f.  physiol.  Chem.,  27;  Salkowski  and  Hahn,  Pfluger's  Arch., 
59;  Salkowski,  ibid.,  63;  v.  Moraczewski,  Zeitschr.  f.  physiol.  Chem.,  20;  Sebelien, 
ibid.,  20. 

3  Salkowski,  1.  c;  Biffi,  Virchow's  Arch.,  152  j  Alexander,  Zeitschr.  f.  physioL 
Chem.,  25. 

3  Zeitschr.  f   physiol.  Chem.,  32. 

*  Ibid.,  25. 


444  MILK. 

O  23.13  per  cent.  Lactalbumin  has  the  properties  of  the  albumins,  and  it 
crystallizes  according  to  Wichmann  1  in  forms  similar  to  ser-  or  ovalbumin. 
It  coagulates,  according  to  the  concentration  and  the  amount  of  salt  in  solu- 
tion, at  72°-84°  C.  It  is  similar  to  seralbumin,  but  differs  from  it  in 
having  a  considerably  lower  specific  rotatory  power:  (a)D  =  —37°. 

The  principle  of  the  preparation  of  lactalbumin  is  the  same  as  for  the 
preparation  of  seralbumin  from  serum.  The  casein  and  the  globulin  are 
removed  by  MgS04  in  substance  and  the  filtrate  treated  as  previously  stated 
(page  153). 

The  occurrence  of  other  proteids,  such  as  proteoses  and  peptones,  in  milk  has 
not  been  positively  proved.  These  bodies  are  easily  produced  as  laboratory 
products  from  the  other  proteids  of  the  milk.  Such  a  laboratory  product  is 
Millon's  and  Comaille's  lactoprotein,  which  is  a  mixture  of  a  little  casein 
with  changed  albumin,  and  proteose,2  which  is  formed  by  chemical  action.  In 
regard  to  opalisin,  see  Human  Milk,  p.  452. 

Milk  also  contains,  according  to  Siegfried,3  a  nucleon  related  to  phos- 
phocarnic  acid,  and  which  yields  fermentation  lactic  acid  (instead  of  para- 
lactic  acid)  and  a  special  carnic  acid,  orylic  acid  (instead  of  muscle  carnic 
acid),  as  cleavage  products.  Lactophosphocarnic  acid  may  be  precipitated 
as  an  iron  combination  from  the  milk  freed  from  casein  and  coagulable 
proteids  as  well  as  from  earthy  phosphates. 

[ilk  also  contains  enzymes  of  various  kinds.  Of  these  we  must  mention  | 
catalase,  oxidases,  and  peroxidases,  which  occur  in  the  various  varieties  of 
milk  in  different  amounts.  Thus,  for  example,  human  milk  contains  small 
amounts  of  oxidases  and  peroxidases,  while  cow's  milk,  on  the  contrary,  is 
richer  in  catalase.  An  enzyme  having  a  saccharifying  action  seems  to  occur 
in  human  milk,  but  is  absent  in  cow 's  milk.  Human  milk,  as  well  as  cow's 
milk,  contains  a  lipase  which  has  the  property  at  least  of  acting  upon  mono- 
butyrin.  /  Babcock  and  Russel  4  have  found  in  these  two  kinds  of  milk, 
a--  well  as  certain  others,  a  proteolytic  enzyme  which  they  call  galactose 
and  which  is  allied  to  trypsin,  but  differs  therefrom  in  that  it  develops 
ammonia  from  milk  even  in  the  early  stages  of  digestion. 

Lactose,  milk-sugar,  C^H^On+H^.  This  sugar,  on  hydrolysis,  can> 
be  split  into  two  hexoses,  dextrose  and  galactose^/  It  yields  mucic  acid, 
besides  other  organic  acids,  by  the  action  of  dilute  nitric  acid.  Levulinic 
acid  is  formed,  besides  formic  acid  and  humin  substances,  by  the  stronger 
action  of  acids.  By  the  action  of  alkalies  amongst  other  products  we  find 
lactic  acid  and  pyrocatechin. 

1  Sebelien,  Zeitschr.  f.  physiol.  Chem.,  9;  Wichmann,  ibid.,  27. 

2  See  Hammarsten,  Maly's  Jahresber.,  6,  13. 

3  Zeitschr.  f.  physiol.  Chem.,  21  and  22. 

4Ceritralbl.  f.  Bakt.  u.  Parasitenk.  (II),  6,  and  Maly's  Jahresber.,  31.  See  also 
Jolles,  Zeitschr.  f.  Biologie,  45,  and  especially  Raudnitz,  Ergebnisse  der  Physiol.,  2, 
Abt.  1. 


LACTOSE.  1  l."> 

f     Milk-sugar  occurs,  as  a  rule,  only  in  milk,  but  it  has  also  been  found  inj 
the  urine  of  pregnant  women  on  stagnation  of  milk,  as  well  as  in  the  urine 
after  partaking  of  large  quantities  of  the  same  sugar^/" 

Lactose,  01  which,  according  to  Tanret,1  there  are  three  modifica- 
tions, occurs  ordinarily  as  colorless  rhombic  crystals  with  1  molecule  of 
water  of  crystallization,  which  is  driven  off  by  slowly  heating  to  100°  C, 
but  more  easily  at  130-140°  C.  At  170°  to  180°  C.  it  is  converted  into  a 
brown  amorphous  mass,  lactocaramel,  C6H10O5.  On  quickly  boiling  down 
a  milk-sugar  solution,  anhydrous  milk-sugar  separates  out./  Milk-sugar  f 
/  dissolves  in  6  parts  cold  or  in  2.5  parts  boiling  water;  it  has  a  faintly  sweet-' 
'  ish  taste^/lt  does  not  dissolve  in  ether  or  absolute  alcohol.  Its  solutions 
are  dextrogyrate.  The  rotatory  power,  which  on  heating  the  solution  to 
100°  C.  becomes  constant,  is  (a)D  =  +  52.5°.y/Milk-sugar  combines  with 
bases;   the  alkali  combinations  are  insoluble  in  alcohok 

Milk-sugar  is  not  fermentable  with  pure  yeast.  It  undergoes,  on  the 
contrary,  alcoholic  fermentation  by  the  action  of  certain  schizomycetes,  and 
according  to  E.  Fischer  2  the  milk-sugar  is  first  split  into  dextrose  and 
galactose  by  an  enzyme,  lactase,  existing  in  the  yeast.  The  preparation  of 
milk- wine,  "kumyss,"  from  mare's  milk  and  "kephir"  from  cow's  milk  is 
based  upon  this  fact.  Other  micro-organisms  also  take  part  in  this  change, 
causing  a  lactic-acid  fermentation  of  the  milk-sugar. 

Lactose  responds  to  the  reactions  of  dextrose,  such  as  Moore's,  1 
Tuo.mmer's,  and  Rubner's,  and  the  bismuth  test.  It  also  reduces  mer- 
curic oxide  in  alkaline  solutions.  After  warming  with  phenylhydrazine 
acetate  it  gives  on  cooling  a  yellow  crystalline  precipitate  of  phenyl- 
lactosazone,  C24H32N409.  It  differs  from  cane-sugar  by  giving  positive 
reactions  with  Moore's  or  Trommer's  and  the  bismuth  test,  and  also  in 
that  it  does  not  darken  when  heated  with  anhydrous  oxalic  acid  to  100°  C. 
It  differs  from  dextrose  and  maltose  by  its  solubility  and  crystalline 
form,  but  especially  by  its  not  fermenting  with  yeast  and  by  yielding  mucic 
acid  with  nitric  acid. 

The  osazone  obtained  with  phenylhydrazine  acetate  which  melts  at  200° 
C.  differs  from  the  other  osazones  by  being  inactive  when  0.2  gram  is  dis- 
solved in  4  c.  c.  of  pyridine  and  6  c.  c.  of  absolute  alcohol  and  viewed 
through  a  layer  10  centimeters  long  (Neuberg  s). 

For  the  preparation  of  milk-sugar  we  make  use  of  the  by-product  in  the 
preparation  of  cheese,  the  sweet  whey.  The  proteid  is  removed  by  coagula- 
tion with  heat,  and  the  filtrate  evaporated  to  a  syrup.  The  crystals  which 
separate  after  a  certain  time  are  recrystallized  from  water  after  decolorizing 
with  animal  charcoal.     A  pure  preparation  may  be  obtained  from  the  com- 


1  Bull.  Soc.  chim.  (3),  13. 

7  Ber.  d.  d.  chem.  Gesellsch.,  27. 

3  Ibid..  22. 


446  MILK. 

mercial  milk-sugar  by  repeated  recrystallization.  The  quantitative  estima- 
tion of  milk-sugar  may  in  part  be  performed  by  the  polaristrobometer  and 
partly  by  means  of  titration  with  Fehling's  solution.  -  Ten  c.  c.  of  Feh- 
ling's solution  correspond  to  0.0676  gram  of  milk-sugar  in  0.5-1.5  percent, 
solution  after  boiling  for  six  minutes.  (In  regard  to  Fehling's  solution  and 
the  titration  of  sugar  see  Chapter  XV.) 

Ritthatjsen  has  found  another  carbohydrate  in  milk  which  is  soluble  in  water, 
non-crystallizable,  which  has  a  faint  reducing  action,  and  which  yields  on  boiling 
with  an  acid  a  body  having  a  greater  reducing  power.  Landwehb  considers- 
this  as  animal  gum,  and  Bechamp  1  as  dextrin. 

The  mineral  bodies  of  milk  will  be  treated  in  connection  with  its  quanti- 
tative composition. 

The  methods  for  the  quantitative  analysis  of  milk  are  very  numerous, 
and  as  they  cannot  all  be  treated  here,  we  will  give  the  chief  points  of  a. 
few  of  the  methods  considered  most  trustworthy  and  most  frequently 
employed. 

X  Tn  determining  the  solids  a  carefully  weighed  quantity  of  milk  is  mixed 
with  an  equal  weight  of  heated  quartz  sand,  fine  glass  powder,  or  asbestos. 
The  evaporation  is  first  done  on  the  water-bath  and  finished  in  a  current  of 
carbon  dioxide  or  hydrogen  not  above  100°  C. 

The  mineral  bodies  are  determined  by  incinerating  the  milk,  using  the  pre- 
cautions mentioned  in  the  text-books.  The  results  obtained  for  the  phosphoric 
acid  are  incorrect  on  account  of  the  burning  of  phosphorized  bodies,  such  as 
casein  and  lecithin.  We  must  therefore,  according  to  Soldner,  subtract 
25  per  cent  from  the  total  phosphoric  acid  found  in  the  milk.  The  quantity 
of  sulphate  in  the  ash  also  depends  on  the  combustion  of  the  proteids. 

In  the  determination  of  the  total  amount  of^jgroteids  Ritthatjsen 's- 
method  is  employed,  namely,  precipitate  the  milk  with  copper  sulphate 
according  to  the  modification  suggested  by  Munk.2  He  precipitates  all 
the  proteids  by  means  of  cupric  hydrate  at  boiling  heat,  and  determines  the 
nitrogen  in  the  precipitate  by  means  of  Kjeldahl's  method.  This  modi- 
fication gives  exacter  results. 

The  older  method  of  Puls  and  Stenberg,  where  the  precipitant  is 
alcohol,  is  too  complicated  and  not  sufficiently  reliable.  Sebelien  has 
su^ested  a  very  good  method.  Three  to  four  grams  of  milk  are  diluted 
with  an  equal  volume  of  water,  a  little  common-salt  solution  added,  and 
precipitated  with  an  excess  of  tannic  acid.  The  precipitate  is  washed  with 
cold  water,  and  then  the  quantity  of  nitrogen  determined  by  Kjeldahl's. 
method.  The  total  nitrogen  found  when  multiplied  by  6.37  (casein  and 
lactalbumin  contain  both  15.7  per  cent  nitrogen)  gives  the  total  quantity  of 
proteids.  This  method,  which  is  readily  performed,  gives  very  good  results. 
I.  Munk  used  this  method  in  the  analysis  of  woman's  milk.  In  this  case 
the  quantity  of  nitrogen  found  must  be  multiplied  by  6.34.  G.  Simon  3  has 
found  that  the  precipitation  with  tannic  acid,  also  with  phosphotungstic 

1  Ritthausen,  Journ.  f.  prakt.  Chem.  (N.  F.),  15;  Landwehr,  foot-note  4,  p.  51; 
B6champ,  Bull.  soc.  chim.  (3),  6. 

2Ritthausen,  1.  c. ;  I.  Munk,  Virchow's  Arch.,  134. 

3  Puis.  Pfliiger's  Arch.,  13;  Stenberg,  Maly's  Jahresber.,  7;  Sebelien,  Zeitschr.  f. 
physiol.  Chem.,  13;  Simon,  ibid.   33. 


METHODS  OF  MILK  ANALYSIS.  447 

acid,  is  the  simplest  and  most  accurate.  Tin-  objection  to  this  and  other 
methods  where  the  proteids  are  precipitated,  is  that  perhaps  other  bodies 
(extractives)  may  be  carried  down  at  the  same  time  (Camerer  and  Sold- 
ner  *).     It  is  undecided  to  what  extent  this  takes  place. 

A  part  of  the  nitrogen  in  the  milk  exists  as  extractives,  and  this  nitrogen  is 
calculated  as  the  difference  between  the  total  nitrogen  and  the  protein  nitrogen. 
According  to  Munk's  analyses  about  T-6  of  the  total  nitrogen  belongs  to  the  ex- 
tractives in  cow's  milk,  and  ^T  in  woman's  milk.  Camerer  and  SoLDNBB  deter- 
mine the  nitrogen  in  the  filtrate  from  the  tannic-acid  precipitate  by  Kjeldaiii.'s 
method,  and  also  according  to  Hufner's  method  (hypobromite).  In  this  way 
they  found  IS  milligrams  of  nitrogen  according  to  Hufner  (urea,  etc.)  in  100 
grams  of  cow's  milk. 

To  determine  the  casein  and  albumim  separately  we  may  make  use  of 
the  method  first  suggested  by  Hoppe-Seyler  and  Tolmatscheff,2  in 
which  the  casein  is  precipitated  by  magnesium  sulphate.  According  to 
Sebelien,  the  milk  is  diluted  with  its  own  volume  by  a  saturated  mag- 
nesium-sulphate solution,  then  saturated  with  the  salt  in  substance,  and 
the  precipitate  then  filtered  and  washed  with  a  saturated  magnesium- 
sulphate  solution.  The  nitrogen  is  determined  in  the  precipitate  by  Kjel- 
dahl's method,  and  the  quantity  of  casein  (+globulin)  determined  by 
multiplying  the  result  by  6\3_2.  The  quantity  of  lactalbumin  may  be  cal- 
culated as  the  difference  between  the  casein  and  the  total  proteids  found. 
The  lactalbumin  may  also  be  precipitated  by  tannic  acid  from  the  filtrate 
from  the  casein  precipitate  containing  MgS04,  after  diluting  with  water,  and 
the  nitrogen  determined  by  Kjeldahl's  method  and  the  result  multi- 
plied by  6.37. 

Schlossmann  3  suggests  an  alum  solution,  which  precipitates  the  casein. 
In  order  to  separate  the  casein  from  the  other  proteids,  the  albumin 
can  be  precipitated  from  the  filtrate  by  tannic  acid.  The  precipitate  is 
used  to  determine  the  nitrogen  by  Kjeldahl's  method.  This  method  has 
recently  been  tested  by  Simon  and  he  recommends  it  highly. 

The  jafc  is  gravimetrically  determined  by  thoroughly  extracting  the 
dried  mf^with  ether,  evaporating  the  ether  from  the  extract,  and  weighing 
the  residue.  The  fat  maybe  determined  by  aerometric  means  by  adding 
alkali  to  the  milk,  shaking  with  ether,  and  determining  the  specific  gravity 
of  the  fat  solution  by  means  of  Soxhlet's  apparatus.  In  determining  the 
amount  of  fat  in  a  large  number  of  samples  the  lactocrit  of  De  Laval  may 
be  used  with  success ,/"TI,he  milk  is  first  mixed  with  an  equal  volume  of  a 
mixture  of  glacial  acetic  and  concentrated  sulphuric  acid,  warmed  7-8 
minutes  on  the  water-bath,  and  the  mixture  poured  in  graduated  tubes,  which 
are  placed  in  the  centrifugal  machine  at  50°  C.  The  height  of  the  layer  of 
fat  gives  its  quantity.  The  numerous  and  very  exact  analyses  of  Nilson4 
have  shown  that  with  milks  containing  small  quantities  of  fat,  below  1.5 
per  cent,  the  older  corrections  are  unnecessary,  and  that  this  method  gives 
excellent  results  if  we  use  lactic  acid  treated  with  5  per  cent  hydrochloric 

1  Zeitschr.  f.  Biologie,  33  and  36. 

2  Hoppe-Seyler,  Med.-chem.  Untersuch.,  272. 
8  Zeitschr.  f.  physiol.  Chem.,  22. 

4  See  Mary's  Jahresber.,  21. 


448  MILK. 

acid  instead  of  the  above  mixture  of  glacial  acetic  acid  and  sulphuric  acid. 
There  are  numerous  other  methods  for  estimating  milk-fat  but  they  cannot 
be  considered  here. 

In  determining  the  milk-sugar  the  proteids  are  first  removed.  For 
this  purpose  we  precipitate  either  with  alcohol,  which  must  be  evaporated 
from  the  filtrate,  or  by  diluting  with  water,  and  removing  the  casein  by 
the  addition  of  a  little  ac  d,  and  the  lactalbumin  by  coagulation  at  boiling 
heat.  The  sugar  is  determined  by  titration  with  Fehling's  or  Knapp's 
solution  (see  Chapter  XV).  The  principle  of  the  titration  is  the  same  as  for 
the  titration  of  sugar  in  the  urine:  10  c.  c.  of  Fehling's  solution  correspond 
to  0.0676  gram  of  milk-sugar;  10  c.  c.  of  Knapp's  solution  correspond  to 
0.0311-0.0310  gram  of  milk-sugar,  when  the  saccharine  liquid  contains  about 
^— 1  per  cent  of  sugar.  In  regard  to  the  modus  operandi  of  the  titration  we 
must  refer  the  reader  to  more  complete  works  and  to  Chapter  XV. 

Instead  of  these  volumetric  determinations  other  methods  of  estima- 
tion, such  as  Allihn's  method,  the  polariscope  method,  and  others,  may 
be  used.  In  calculating  the  analysis  or  in  determining  the  solids  it  is  of 
importance  to  remember,  as  suggested  by  Camerer  and  Soldner,  that  the 
milk-sugar  in  the  residue  is  anhydrous.  Many  other  methods  for  determin- 
ing the  milk-sugar  have  been  suggested  and  recommended. 

The  quantitative  composition  of  cow's  milk  is  naturally  very  variable. 
The  average  obtained  by  Konig  x  is  as  follows  in  1000  parts : 

Water.  Solids.  Casein.         Albumin.  Fats.  Sugar.  Salts. 

871.7  128.3  30.2  5.3  36.9  48.8  7.1 


35.5 

The  quantity  of  mineral  bodies  in  1000  parts  of  cow's  milk  is,  according 
to  the  analyses  of  Soldner,  as  follows:  K30  1.72,  Na20  0.51,  CaO  1.98, 
MgO  0.20,  P205  1.82  (after  correction  for  the  pseudonuclein) ,  CI  0.98  grams. 
Sunge  2  found"  0.0035  gram  Fe203.  According  to  Soldner,  the  K,  Na, 
and  CI  are  found  in  the  same  quantities  in  whole  milk  as  in  milk-serum. 
Of  the  total  phosphoric  acid  36-56  per  cent  and  of  the  lime  53-72  per  cent 
is  not  in  solution.  A  part  of  this  lime  is  combined  with  the  casein;  the 
remainder  is  found  united  with  the  phosphoric  acid  as  a  mixture  of  clical- 
cium  and  tricalcium  phosphate,  which  is  kept  dissolved  or  suspended  by  the 
casein.  The  bases  are  in  excess  of  the  mineral  acids  in  the  milk-serum. 
The  excess  of  the  first  is  combined  with  organic  acids,  which  correspond 
to  2.5  p.  m.  citric_acid  (Soldner). 

The  gasgs  of  the  milk  consist  chiefly  of  COg,  besides  a  little  N  and  traces 
of  Qj  Pfluger  3  found  10  vols,  per  cent  C02  and  0.6  vol.  per  cent  N 
calculated  at  0°  C.  and  760  mm.   pressure. 

The  variation  in  the  composition  of  cow's  milk  depends  on  several 
circumstances. 

1  Chemie  der  menschlichen  Nahrungs-  und  Genussmittel,  4.  Aufl. 

2  Zeitschr.  f.  Biologie,  10. 

3  Pfluger 's  Arch.,  2. 


COLOSTRUM.  449 

The  colostrum,  or  the  milk  which  is  secreted  before  calving  and  in  the 
first  few  days  after,  is  yellowish,  sometimes  alkaline,  but  often  acid,  of 
higher  specific  gravity,  1.046-1.080,  and  richer  in  solids  than  ordinary 
milk.  The  colostrum  contains,  besides  fat^globules,  an  abundance  of 
colostrum-corpuscles — nucleated  granular  cells  0.005-0.025  mm.  in  diam- 
eter with  abundant  fat-granules  and  fat-globules.  The  fat  of  colostrum 
has  a  somewhat  higher  melting-point  and  is  poorer  in  volatile  fatty  acids 
than  the  fat  from  ordinary  milk  (Nilson  l).  The  quantity  of  cholesterin  and 
lecithin  is  generally  greater.  The  most  apparent  difference  between  it 
and  ordinary  milk  is  that  colostrum  coagulates  on  heating  to  boiling  because 
of  the  absolute  and  relatively  greater  quantities  of  globulin  and  albumin 
it  contains.2  The  composition  of  colostrum  is  very  variable.  Koxig  gives 
as  average  the  following  figures  in  1000  parts:  ■  —  J 

Water.  Solids.  Casein.    Albumin  and  Globulin.      Fat.  Sugar.  Salts. 

746.7  253.3  40.4  136.0  35.9  26.7  15.6 

The  influence  which  food  exercises  upon  the  composition  of  milk  will 
be  discussed  in  connection  with  the  chemistry  of  the  milk  secretion. 

In  the  following  table  is  given  the  average  composition  of  skimmed  milk  and 
certain  other  preparations  of  milk: 

Water.  Proteids.  Fat.  Sugar.  Lactic  Acid.  Salts. 

Skimmedmilk 906.6  31.1  7.4  47.5         ...         7.4 

Cream 655.1  36.1  267.5  35.2         ...         6.1 

Buttermilk 902.7  40.6  9.3  37.3         3.4         6.7 

Whey 932.4  8.5  2.3  47.0         3.3         6.5 

Kumyss  and  kephir  are  obtained,  as  above  stated,  by  the  alcoholic  and  lactic- 
acid  fermentation  of  the  milk-sugar,  the  first  from  mare's  milk  and  the  last  from 
cow's  milk.  Large  quantities  of  carbon  dioxide  are  formed  thereby,  and  besides 
the  proteid  bodies  of  the  milk  are  partly  converted  into  proteoses  and  peptones, 
which  increase  the  digestibility.  The  quantity  of  lactic  acid  in  these  preparations 
may  be  about  10-20  p.  m.     The  quantity  of  alcohol  varies  from  10  to  35  p.  m. 

Milk  of  other  Animals.  Goat's  milk  has  a  more  yellowish  color  and  another 
more  specific  odor  than  cow's  milk.  The  coagulum  obtained  by  acid  or  rennet 
is  more  solid  and  is  harder  than  that  from  cow's  milk.  Sheep's  milk  is  similar 
to  goat's  milk,  but  has  a  higher  specific  gravity  and  contains  a  greater  amount 
of  solids. 

Mare's  milk  is  alkaline  and  contains  a  casein  which  is  not  precipitated  by 
acids  in  lumps  or  solid  masses,  but,  like  the  casein  from  woman's  milk,  in  fine 
flakes.  This  casein  is  only  incompletely  precipitated  by  rennet,  and  it  is  very 
similar  also  in  other  respects  to  the  casein  of  human  milk.  According  to  Rett.,3 
the  casein  from  mare's  and  cow's  milk  is  the  same,  and  the  different  behavior 
of  the  two  varieties  of  milk  is  due  to  different  amounts  of  salts  and  to  a  different 
relation  between  the  casein  and  the  albumin.  The  milk  of  the  ass  is  claimed 
by  older  authorities  to  be  similar  to  human  milk,  but  Schlossmann  finds  it  con- 

1  Nilson,  1.  c. 

2 See  Sebelien,  Maly's  Jahresber.,  18,  and  Tiemann,  Zeitschr.  f.  phvsioL  Chem., 
25.     See  also  Simon,  ibid.,  33. 

5  Studien  iiber  die  Eiweissstoffe  des  Kumys  und  Kefirs.  St.  Petersburg,  1886. 
(Ricker) 


Solids. 

Proteids. 

Fat. 

Sugar. 

Salts. 

245.6 

99.1 

95.7 

31.9 

7.3 

183.7 

90.8 

33.3 

49.1 

5.8 

130.9 

36.9 

40.9 

44.5 

8.6 

165.0 

57.4 

61.4 

39.6 

6.6 

128.3 

35.5 

36.9 

48.8 

7.1 

99.4 

18.9 

10.9 

66.5 

3.1 

100.0 

21.0 

13.0 

63.0 

3.0 

167.3 

60.9 

64.4 

40.4 

10.6 

321.5 

30.9 

195.7 

88.4 

6.5 

513.3 

437.6 

.... 

4.6 

Human 

Milk. 

450  MILK. 

siderably  poorer  in  fat.  The  researches  of  Ellenberger  give  similar  results, 
and  show  great  similarity  between  ass's  milk  and  human  milk.  The  average 
results  were  15  p.  m.  proteid  with  5.3  p.  m.  albumin  and  9.4  p.  m.  casein.  This 
latter,  like  human  casein,  does  not  yield  any  pseudonuclein  on  pepsin  digestion. 
The  quantity  of  nucleon  was  about  the  same  as  in  woman's  milk.  The  quantity 
of  fat  was  15  p.  m.,  and  the  sugar  was  50-60  p.  m.  Reindeer  milk  characterizes 
itself,  according  to  Werenskiold,1  by  being  very  rich  in  fat,  144.6-197.3  p.  m., 
and  casein,  80.6-86.9  p.  m. 

The  milk  of  carnivora  (the  bitch  and  cat)  is  acid  in  reaction  and  very  rich 
in  solids.  The  composition  of  the  milk  of  these  animals  varies  with  the  compo- 
sition of  the  food. 

To  illustrate  the  composition  of  the  milk  of  other  animals  the  following  figures, 
the  compilation  of  Konig,  are  given.  As  the  milk  of  each  kind  of  animal  may 
have  a  variable  composition,  these  figures  should  only  be  considered  as  examples 
of  the  composition  of  milk  of  various  kinds : 2 

Milk  of  the  Water. 

Dog 754.4 

Cat 816.3 

Goat 869 . 1 

Sheep 835.0 

Cow 871.7 

Horse 900.6 

Ass 900.0 

Pig 823.7 

Elephant 678.5 

Dolphin 486.7 


Woman's  milk  is  amphoteric  in  reaction.  According  to  Courant  its 
reaction  is  relatively  more  alkaline  than  cow's  milk,  but  has  nevertheless  a 
lower  absolute  reaction  for  alkalinity  as  well  as  acidity  ,/Courant  found  be- 
f  tween  the  tenth  day  and  the  fourteenth  month  after  confinement  practically 
constant  results.  The  alkalinity,  as  well  as  the  acidity,  was  a  little  lower 
than  in  childbed/^  Ohe  hundred  c.  c.  of  the  milk  had  the  same  average 
alkalinity  as  10.8  c.  c.  N/10  caustic  soda,  and  the  same  acidity  as  3.6  c.  c. 
N/10  acid.  The  relationship  between  the  alkalinity  and  the  acidity  in 
woman's  milk  was  as  3:1,  and  in  cow's  milk  as  2.1:1. 

/  Human  milk  also  contains  fewer  fat-globules  than  cow 's  milk,  but  they 
are  larger  in  size.  The  specific  gravity  of  woman's  milk  varies  between 
1026  and  1036,  generally  between  1028  and  1034.  It  is  highest  in  well- 
fed  and  lowest  in  poorly  fed  women. J  The  freezing-point  is  lowered  on 
an  average  0.589°  C.  and  the  molecular  concentration  is  0.318. 

The  fat  of  woman's  milk  has  been  investigated  by  Ruppel.  It  forms  a 
yellowish-white  mass,  similar  to  ordinary  butter,  having  a  specific  gravity 
of  0.966  at  15°  C.  It  melts  at  34.0°  C.  and  solidifies  at  20.2°  C.  The  follow- 
ing fatty  acids  can  be  obtained  from  the  fat,  namely,  butyric,  caproic, 

•Schlossmann,  Zeitschr.  f.  physiol.  Chem.,  22;  Ellenberger,  Arch.  f.  (Anat.  u.) 
Physiol.,  1899  and  1902;  Werenskiold,  Maly's  Jahresber.,  25. 

2  Details  in  regard  to  the  milk  of  different  animals  may  be  found  in  Proscher, 
Zeitschr.  f.  physiol.  Chem.,  24;  Abderhalden,  ibid.,  27. 


HUMAN   MILK.  451 

capric,  myristic,  palmitic,  stearic,  and  oleic  acids.  The  fat  from  woman's 
milk  is,  according  to  Ruppel  and  Laves,1  relatively  poor  in  volatile  fatty 
acids.  The  non-volatile  fatty  acids  consist  of  one-half  oleic  acid,  while 
anions  the  solid  fatty  acids  myristic  and  palmitic  acids  are  found  to  a 
greater  extent  than  stearic  acid. 

f~  The  essential  qualitative  difference  between  woman's  and  cow's  milk 
■'  seems  to  lie  in  the  proteids  or  in  the  more  accurately  determined  casein. 
A  number  of  older  and  younger  investigators  2  claim  that  the  casein  from 
woman's  milk  has  other  properties  than  that  from  cow's  milk.  The  essen- 
tial differences  are  the  following:  The  casein  from  woman's  milk  is  precipi- 
tated with  greater  difficulty  with  acids  or  salts;  it  does  not  coagulate  regu- 
larly in  the  milk  after  the  addition  of  rennet;  it  may  be  precipitated  by 
gastric  juice,  but  dissolves  completely  and  easily  in  an  excess  of  the  same; 
the  casein  precipitate  produced  by  an  acid  is  more  easily  soluble  in  an  excess 
of  the  acid;  and  lastly,  the  clot  formed  from  the  casein  of  woman's  milk 
does  not  appear  in  such  large  and  coarse  masses  as  the  casein  from  cow's 
milk,  but  is  more  loose  and  flocculent.  This  last-mentioned  fact  is  of  great 
importance,  since  it  explains  the  generally  admitted  fact  of  the  easy 
digestibility  of  the  casein  from  woman's  milk.  We  are  not  clear  as  to 
this  difference  between  the  digestibility  of  the  cow's  casein  and  human 
casein,  as  the  first  seems  to  be  utilized  in  the  intestinal  tract  of  the  infant 
to  the  same  extent  as  human  casein  (P.  Muller,  Rubxer  and  Heubxer  3)., 
The  question  as  to  whether  the  above-mentioned  differences  depericTon 
a  decided  difference  in  the  two  caseins  or  only  on  an  unequal  relationship 
between  the  casein  and  the  salts  in  the  two  kinds  of  milk,  or  upon  other 
circumstances,  has  not  been  decided  as  yet.  According  to  Szoxtagh,  the 
casein  from  human  milk  does  not  yield  any  pseudonuclein  on  peptic  diges- 
tion, and  hence  it  cannot  be  a  nucleoalbumin.  Wr6blewsky  has  recently 
arrived  at  the  same  results,  and  also  has  found  that  the  two  caseins  had 
a  different  composition.  He  found  the  following  for  the  composition  of 
casein  from  woman's  milk:  C  52.24,  H  7.32,  N  14.97,  P  0.68,  S  1.117  per  cent. 
According  to  Kobrak,4  woman's  casein  yields  some  pseudonuclein,  and 
with  repeated  solution  in  alkali  and  precipitation  by  an  acid,  it  becomes 
more  and  more  like  cow's  casein.     He  therefore  suggests  the  possibility 


1  Ruppel,  Zeitschr.  f.  Biologie,  31;  Laves,  Zeitschr.  f.  physiol.  Chem.,  19. 

1  See  Biedert,  Untersuchungen  iiber  die  chemischen  Unterschiede  der  Menschen- 
und  Kuhmilch  (Stuttgart),  1884;  Langgaard,  Virchow's  Arch.,  66;  Makris,  Studien 
iiber  die  Eiweisskorper  der  Frauen-  und  Kuhmilch.      Inaug.-Diss.      Strasburg,   1S76. 

3  Muller,  Zeitschr.  f.  Biologie,  39;   Rubner  and  Heubner,  ibid.,  37. 

4Szontagh,  Maly's  Jahresber.,  22;  Wroblewsky,  "Beitriige  zur  Kenntnisse  des 
Frauenkaseins"  (Inaug.-Diss.,  Bern,  1894),  and  "Ein  neuer  eiweissartiger  Bestand- 
theil  der  Milch,"  Anzeiger  der  Akad.  d.  Wiss.  in  Krakau,  1898;  Kobrak,  Pfliiger's 
Arch.,  80. 


452  MILK. 

that  woman's  casein  is  a  compound  between  a  nucleoalbumin  and  a  basic 
proteid. 

Woman's  milk  also  contains  lactalbumin,  besides  the  casein,  and  a 
protein  substance,  very  rich  in  sulphur  (4.7  per  cent)  and  relatively  poor 
in  carbon,  which  Wroblewsky  calls  opalisin.  The  statements  as  to  the 
occurrence  of  proteoses  and  peptone  are  disputed  as  in  many  other  cases. 
No  positive  proof  as  to  the  occurrence  of  proteoses  and  peptone  in  fresh 
milk  has  been  given. 

Even  after  those  differences  are  eliminated  which  depend  on  the  imper- 
fect analytical  methods  employed,  the  quantitative  composition  of  woman's 
milk  is  variable  to  such  an  extent  that  it  is  impossible  to  give  any  average 
results.  The  recent  analyses,  especially  those  made  on  a  large  number 
of  samples  by  Pfeiffer,  Adriance,  Camerer  and  Soldner,1  have  posi- 
tively shown  that  woman's  milk  is  essentially  poorer  in  proteids  but  richer 
in  sugar  than  cow's  milk.  The  quantity  of  proteid  varies  between  10-20 
p  m.,  often  amounting  to  only  15-17  p.  m.  or  less,  and  is  dependent  upon 
the  length  of  lactation  (see  below). /^The  quantity  of  fat  also  varies  con- 
siderably, but  ordinarily  amounts  to  30-40  p.  m.  The  quantity  of  sugar 
should  not  be  below  50  p.  m.,  but  may  rise  to  even  80  p.  m.  About! 
60  p.  m.  may  be  considered  as  an  average,  but  it  should  be  borne  in 
mind  that  the  quantity  of  sugar  is  also  dependent  upon  the  length  of 
lactation,  as  it  increases  with  durationy^The  amount  of  mineral  bodies 
varies  between  2  and  4  p.  m. 

From  a  quantitative  standpoint,  the  most  essential  differences  between 
woman's  and  cow's  milk  are  as  follows:  As  compared  with  the  quantity 
of  albumin,  the  quantity  of  casein  is  not  only  absolutely  but  also  relatively 
smaller  in  woman's  milk  than  in  cow's  milk,  while  the  latter  is  poorer  in 
milk-sugar.  Human  milk  is  richer  in  lecithin  at  least  relative  to  the  amount 
of  proteid.  Burow  found  0  49-0.58  p.  m.  lecithin  in  cow's  mi'k  and  0.58 
p.  m.  in  woman's  milk,  which  corresponds  to  1.40  per  cent  for  the  first 
milk  and  3.05  per  cent  for  the  second,  calculated  on  the  percentage  of 
proteid.  The  quantity  of  nucleon  is  greater  in  woman's  milk.  According 
to  WiTTMAACK  cow's  milk  contains  0.566  p.  m.  nucleon,  and  woman's 
milk  1.24  p.  m.  Siegfried  2  finds  that  the  nucleon  phosphorus  amounts 
to  6.0  per  cent  of  the  total  phosphorus  in  cow's  milk  and  41.5  per  cent  in 
woman's  milk,  and  also   that  in  human  milk  the  phosphorus  is  nearly 

1  Pfeiffer,  Jahrb.  f.  Kinderheilkunde,  20;  also  Maly's  Jahresber.,  13;  V.  Adriance 
and  J.  Adriance,  A  Clinical  Report  of  the  Chemical  Examination,  etc.,  Archives  of 
Pediatrics,  1897;  Camerer  and  Soldner,  Zeitschr.  f.  Biologie,  33  and  36.  In  regard 
to  the  composition  of  woman's  milk,  see  also  Biel,  Maly's  Jahresber.,  4;  Christenn, 
ibid.,  7;  Mendes  de  Leon,  ibid.,  12;  Gerber,  Bull.  soc.  Chim.,  23;  Tolmatscheff,  Hoppe- 
Seyler's  Med.-chem.  Untersuch.,  272. 

2  Burow,  Zeitschr.  f.  physiol.  Chem.,  30;   Wittmaack,  ibid.,  22;   Siegfried,  ibid.,  22. 


HUMAN  MILK.  453 

entirely  in  organic  combination.  Woman's  milk  is  poorer  in  mineral 
bodies,  especially  lime,  and  it  contains  only  one-sixth  of  the  quantity  of 
lime  as  compared  with  cow's  milk.  Human  milk  is  claimed  to  be  also 
poorer  in  citric  acid  (Scheibe1),  although  this  is  not  an  essential  difference. 

Another  difference  between  woman's  milk  and  other  varieties  of  milk  is  Umi- 
koff's  reaction,  which  seems  to  depend  upon  the  quantitative  composition, 
especially  the  relation  between  the  milk-sugar,  citric  acid,  lime,  and  iron  (Sieber.2) 
This  reaction  consists  in  treating  5  c.  c.  of  woman's  milk  with  2.5  c.  c.  ammonia 
(10  per  cent)  and  heating  to  60°  C.  for  15-20  minutes,  when  the  mixture  becomes 
violet-red.     Cow's  milk  gives  a  yellowish-brown  color  when  thus  treated. 

According  to  Rubner  woman's  milk  contains  about  3  p.  m.  soaps,  but  this 
could  not  be  substantiated  by  Camerer  and  Soldner.  According  to  them 
woman 's  milk  contains  no  soaps,  or  at  least  only  very  small  amounts.  They  also 
found  the  quantity  of  urea  nitrogen  in  woman's  milk  to  be  0.11-0.12  p.m., 
although  Schondorff  3  found  nearly  twice  this  amount,  namely  0.23  p.  m. 

In  regard  to  the  quantity  of  mineral  bodies  in  woman's  milk  we  have 
the  analyses  of  several  investigators,  especially  of  Bunge  (analyses  A  and 
B)  and  of  Soldner  and  Camerer  (analysis  C  4).  Bunge  analyzed  the 
milk  of  a  woman,  fourteen  days  after  delivery,  whose  diet  contained 
very  little  common  salt  for  four  days  previous  to  the  analysis  (A),  and 
again  three  days  later  after  a  daily  addition  of  30  grams  of  NaCl  to  the  food 
(B).    The  figures  are  in  1000  parts  of  the  milk: 

A 

K20 0.780 

Na20 0.232 

CaO 0.328 

MgO 0.064 

Fe203 0.004 

P2Os 0.473 

CI 0.438 

The  relationship  of  the  two  bodies,  potassium  and  sodium,  to  each  other 
may,  according  to  Bunge,  vary  considerably  (1.3-4.4  equivalents  of  potash  to 
1  of  soda)  VT^y  the  addition  of  salt  to  the  food  the  quantity  of  sodium  and 
chlorine  in  the  milk  increases,  while  the  quantity  of  potassium  decreases. 
De  Lange  found  more  Na  than  K  in  the  milk  at  the  beginning  of  lacta- 
tion. Jolles  and  Friedjunq  r'  found  on  an  average  5.0  milligrams  of  iron 
per  liter  of  woman's  milk. 

The  gases  of  woman's  milk  have  been  investigated  by  Kulz.8    He  found 

'Maly's  Jahresber.,  21. 
'Zeitschr.  f.  physiol.  Chem.,  30. 

3  Rubner,  Zeitschr.  f.  Biologie,  36 j  Camerer  and  Soldner,  ibid.,  39  j  Schondorff, 
Pfliiger's  Arch.,  SI. 

4  Bunge,  Zeitschr.  f.  Biologie,  10;  Camerer  and  Soldner,  ibid.,  39  and  44. 

5De  Lange,  Maly's  Jahresber.,  27;  Jolles  and  Friedjung,  Arch.  f.  exp.  Path.  u. 
Pharm.,  4(5. 

8  Zeitschr.  f.  Biologie,  32. 


B 

C 

0.703 

0.884 

0.257 

0.357 

0.343 

0.378 

0.065 

0.053 

0.006 

0.002 

0.469 

0.310 

0.445 

0.591 

454  MILK. 

1.07-1.44  c.  c.  of  oxygen,  2.35-2.87  c.  c.  of  carbon  dioxide,  and  3.37-3.81 
c.  c.  of  nitrogen  in  100  c.  c.  of  milk. 

The  proper  treatment  of  cow's  milk  by  diluting  it  with,  water  and  by 
certain  additions  in  order  to  render  it  a  proper  substitute  for  woman's  milk 
in  the  nourishment  of  children  cannot  be  determined  before  the  difference 
in  the  proteid  bodies  of  these  two  kinds  of  milk  has  been  completely 
studied. 

s  The  colostrum  has  a  higher  specific  gravity,  1.040-1.060,  a  greater  quan- 
tity of  coagulable  proteids,  and  a  deeper  yellow  color  than  ordinary  woman's 
milk.  Even  a  few  days  after  delivery  the  color  becomes  less  yellow,  the 
quantity  of  albumin  less,  and  the  number  of  colostrum-corpuscles  dimin- 
ishes. 

We  have  the  older  analyses  of  Cleatm  l  and  the  recent  investigations  of 
Pfelffer,  V.  and  J.  Adriaxce,  Camerer  and  Soldxer  on  the  changes  in 
the  composition  of  milk  after  deliverv/"lt  follows,  as  a  unanimous  result 
from  these  investigations,  that  the  quantity  of  proteid,  which  amounts 
to  more  the  first  two  days,  sometimes  to  more  than  30  p.  m.  at  first, 
rather  quickly  and  then  more  gradually  diminishes  as  long  as  the  lacta- 
tion continues,  so  that  in  the  third  week  it  equals  about  10-18  p.  m. 
Like  the  protein  substances  so  do  the  mineral  bodies  gradually  decrease. 
The  quantity  of  fat  shows  no  regular  or  constant  variation  during  lactation, 
while  the  lactose,  especially  according  to  the  observations  of  V.  and  J. 
Adriance  (120  analyses),  increases  rather  quickly  the  first  days  and  then 
only  slowly  until  the  end  of  lactation.  The  analyses  of  Pfeiffer,  Camerer 
and  Soldxer  also  show  an  increase  in  the  quantity  of  milk-sugai^/ 

The  two  mammary  glands  of  the  same  woman  may  yield  somewhat  different 
milk,  as  shown  by  Sourdat  and  later  by  Bruxxer.2  Likewise  the  different 
portions  of  milk  from  the  same  milking  may  have  varying  composition.  The 
first  portions  are  always  poorer  in  fat. 

According  to  l  Heritier,  \  erxois  and  Becquerel,  the  milk  of  blondes  con- 
tains less  casein  thimthat  of  brunettes,  a  difference  which  Tolmatscheff  3  could 
not  substantiatey^Women  of  delicate  constitutions  yield  a  milk  richer  in  solids, 
especiallv  in  casein,  than  women  with  strong  constitutions  (Y.  and  B.)^/ 

According  to  Verxois  and  Becquerel,  the  age  of  the  woman  has  an  effect  on 
the  composition  of  the  milk,  so  that  we  find  a  greater  quantity  of  proteids  and 
fat  in  women  1.5-29  years. old  and  a  smaller  quantity  of  sugar.  The  smallest 
quantitv  of  proteids  and  the  greatest  quantity  of  sugar  are  found  at  20  or  from 
25-30  vears  of  age.  According  to  V.  and  B.,  the  milk  with  the  first-born  is  richer 
m  water — with  a  proportionate  diminution  of  casein,  sugar,  and  fat — than  after 
several  deliveries. 

The  influence  of  menstruation  seems  to  slightly  diminish  the  milk-sugar  and  to 
considerablv  increase  the  fat  and  casein  (V.  and  B.). 


1  See  Hoppe-Seyler,  Physiol.  Chem.,  734. 

1  Sourdat,  Compt.  rend.,  71;  Brunner,  Pfluger's  Arch.,  7. 

THeritier,  cited  from  Hoppe-Seyler,  Physiol.  Chem.,  738;  Vernois  and  Bec- 
querel, Du  lait  chez  la  femme  dans  l'etat  de  sante,  etc.  (Paris,  1853) j  Tolmatscheff, 
Hoppe-Seyler,  Med.-chem.  Untersuch.,  272. 


MILK  AND  NEW-BORN   YOUNG.  455 

Witch's  milk  is  the  secretion  of  the  mammary  glands  of  new-born  children  of 
both  sexes  immediately  after  birth.  This  secretion  has  from  a  qualitative  stand- 
point the  same  constitution  as  milk,  but  may  show  important  differences  and 
variations  from  a  quantitative  point  of  view.  Schlossberger  and  Hauff, 
Gxtblbb  and  Quevenne,  and  v.  Genser  l  have  made  analyses  of  this  milk  and 
give  the  following  results:  10.5-28  p.  m  proteids,  8.2-14.6  p.  m.  fat,  and  9-00  p.  m. 
sugar. 

As  milk  is  the  only  form  of  nourishment  during  a  certain  period  of  the 
life  of  man  and  mammals,  it  must  contain  all  the  nutriment  necessary  for 
life.  This  fact  is  shown  by  the  milk  containing  representatives  of 
the  three  chief  groups  of  organic  nutritive  substances — proteids,  carbohy- 
drates, and  fat;  and  all  milk  seems  to  contain  without  doubt  also  some 
lecithin  and  nucleoli.  The  mineral  bodies  in  milk  must  also  occur  in  proper 
proportions,  and  on  this  point  the  experiments  of  Bunge  on  dogs  are  of 
special  interest.  He  found  that  the  mineral  bodies  of  the  milk  occur  in 
about  the  same  relative  proportion  as  they  do  in  the  body  of  the  sucking 
animal.  Bunge  2  found  in  1000  parts  of  the  ash  the  following  results 
(A  represents  results  from  the  new-born  dog,  and  B  the  milk  from  the  bitch) : 

A.  B. 

K20 114.2  149.8 

Na20 106.4  88.0 

CaO 295.2  272.4 

MgO 18.2  15.4 

FeA, 7.2  1.2 

P,C)5 394.2  342.2 

CI 83.5  169.0 

Bunge  explains  the  fact  that  the  milk-ash  is  richer  in  potash  and  poorer 
in  soda  than  the  new-born  animal  by  saying  that  in  the  growing  animal  the 
ash  of  the  muscles  rich  in  potash  relatively  increases  and  the  cartilage  rich 
in  soda  relatively  decreases.  In  regard  to  the  amount  of  iron  we  find  an 
unexpected  condition,  the  ash  of  the  new-born  animal  containing  six  times 
as  much  as  the  milk-ash.  This  condition  Bunge  explains  by  the  fact 
founded  on  his  and  Zalesky's  experiments,  that  the  quantity  of  iron  in  the 
entire  organism  is  highest  at  birth.  The  new-born  has  therefore  its  own 
supply  of  iron  for  the  growth  of  its  organs  even  at  birth^     / 

The  investigations  of  Hugounenq,  de  Lange,  Camerer  and  Soldner  3 
have  shown  that  in  man  the  conditions  are  different  from  those  in  animals, 
as  the  ash  of  the  child  has  an  entirely  different  composition  as  compared  to 
the  milk.     As  an  example  the  following  analyses  are  given  (of  Camerer  and 


1  Schlossberger  and  Hauff,  Annal.  d.  Chem.  u.  Pharm.,  96;  Gubler  and  Quevenne, 
cited  from  Hoppe-Seyler 's  Physiol.  Chem.,  723;  v.  Genser,  ibid. 

1  Zeitschr.  f.  physiol.  Chem.,  13. 

'Hugounenq,  Compt.  rend.,  128;  de  Lange,  Zeitschr.  f.  Biologie,  40;  Camerer 
and  Soldner,  ibid.,  39,  40.  and  44. 


•±56  MILK. 


Soldner).     (A,  the  ash  of  the  sucking  infant,  and  B,  the  ash  of  the  milk.) 
The  results  are  in  1000  parts  of  the  ash. 


K20. 
Na,0 
CaO. 
MgO. 
Fe203 

p2o5. 

CI... 


A. 

B. 

78 

314 

91 

119 

361 

164 

9 

26 

8 

6 

389 

135 

77 

200 

We  cannot  therefore  state  as  a  definite  fact  that  the  composition  of  the 

ash  of  the  sucking  young  and  the  ash  of  the  corresponding  milk  coincide. 
Bunge  *  nevertheless  claims  that  the  composition  of  the  ash  of  the  sucking 
young  of  various  mammals  is  nearly  the  same,  but  that  the  ash  of  the  milk 
differs  from  the  ash  of  the  young  in  so  far  as  the  slower  the  young  grows 
the  richer  it  is  in  alkali  *  hlorides  and  relatively  poorer  in  phosphates  and 
lime-salts.  The  constituents  of  the  ash  have  two  functions  to  perform, 
namely,  the  building  up  of  the  tissues  and  secondly  the  preparation  of  the 
excreta,  especially  the  urine.  The  faster  the  young  grows  the  more  is  the 
first  in  evidence,  while  the  slower  it  develops,  the  second  is  prominent.  ^S 
"^The  quantity  of  mineral  bodies  in  the  milk,  and  especially  the  amount 
of  lime  and  phosphoric  acid,  as  shown  by  Bunge  and  Proscher  and  Pages, 
stands  in  close  relationship  to  the  rapidity  of  growth,  because  the  amount 
of  these  mineral  constituents  in  the  milk  is  greater  in  animals  which  grow 
and  develop  quickly  than  in  those  which  grow  only  slowly.  A  similar 
relationship  exists  also,  as  shown  by  the  researches  of  Proscher,  and  espe- 
cially of  Abderhalden,2  between  the  quantity  of  proteid  in  the  milk  and 
the  rapidity  of  development  of  the  sucking  young.     The  amount  of  proteid  / 

st 

LS. 

"From  these  we  learn  that  in  human  beings  as  well  as  in  animals  an 
insufficient  diet  decreases  the  quantity  of  milk  and  the  quantity  of  solids, 
while  abundant  food  increases  both.  From  the  observations  of  De- 
caisxe  3  on  nursing  women  during  the  siege  of  Paris  in  1871,  the  amount 
of  casein,  fat,  sugar,  and  salts,  but  especially  the  fat,  was  found  to 
decrease  with  insufficient  food,  while  the  quantity  of  lactalbumin  was  found 
to  be  somewhat  increased.  Food  rich  in  proteids  increases  the  quantity  of 
fflilk.  and  also  the  solids  contained,  especially  the  fat,  according  to  most 

'Bunge,   "Die    zunehmende  Unfahigkeit    der  Frauen   ihre    Kinder  zu  still  en," 
Munchen,  1900,  cited  by  Camerer,  Zeitschr.  f.  Biologie,  40. 

2  Proscher,  Zeitschr.  f.  physiol.  Chem.,  24;    Abderhalden,  ibid.,  27;    Pages,  Arch, 
de  Physiol.  (5),  7. 

3  Cited  from  Hoppe-Seyler,  1.  c,  739. 


is  greater  in  the  milk  the  quicker  the  animal  develops. 

The  influence  of  the  food  on  the  composition  of  the  milk  is  of  interest  ^ 
from  many  points  of  view  and  has  been  the  subject  of  many  investigations. 


CHEMISTRY  OF  MILK  SECRETION.  457 

statements.  The  quantity  of  sugar  in  woman's  milk  is  found  by  certain 
|  investigators  to  be  increased  after  food  rich  in  proteids,  while  others  claim  it 
is  diminished.  A  diet  rich  in  fat  may,  as  the  recent  researches  of  SOXHLET 
and  many  others  '  have  shown,  cause  a  marked  increase  in  the  fat  of  the 
milk  when  the  fat  partaken  is  in  a  readily  digestible  and  assimilable  form.  ] 
The  presence  of  large  quantities  of  carbohydrates  in  the  food  seems  to  cause  | 
no  constant,  direct  action  on  the  quantity  of  the  milk-constituent^^Tn 
carnivora,  as  shown  by  Ssuhotix,3  the  secretion  of  milk-sugar  proceeds 
uninterruptedly  on  a  diet  consisting  exclusively  of  lean  meat.  Watery 
food  gives  a  milk  containing  an  excess  of  water  and  having  little  value.  In 
the  milk  from  cows  which  were  fed  on  distillers'  grain  Commaille  4  found 
906.5  p.  m.  water,  26.4  p.  m.  casein,  4.3  p.  m.  albumin,  18.2  p.  m.  fat,  and 
33.8  p.  m.  sugar.  Such  milk  has  sometimes  a  peculiar  sharp  after-taste, 
although  not  always.3 

mittry  of  Milk-secretion.  That  the  constituents  which  occur  actu- 
ally dissolved  in  milk  pass  into  the  secretion  not  alone  by  filtration  or 
diffusion,  but  more  likely  are  secreted  by  a  specific  secretory  activity  of  the 
glandular  elements,  is  shown  by  the  fact  that  milk-sugar,  which  is  not 
found  in  the  blood,  is  to  all  appearances  formed  in  the  glands  themselves 
A  further  proof  lies  in  the  fact  that  the  lactalbumin  is  not  identical  with 
seralbumin;  and  lastly,  as  Buxge  6  has  shown,  the  mineral  bodies  secreted 
by  the  milk  are  in  quite  different  proportions  from  those  in  the  blood 
[   serum. 

Little  is  known  in  regard  to  the  formation  and  secretion  of  the  specific 
constituents  of  milk.  The  older  theory,  that  the  casein  was  produced  from 
the  lactalbumin  by  the  action  of  an  enzyme,  is  incorrect  and  originated 
probably  from  mistaking  an  alkali-albuminate  for  casein.  Better  founded 
is  the  statement  that  the  casein  originates  from  the  protoplasm  of  the 
glaiui-cells^/There  does  not  seem  to  be  any  doubt  that  the  protoplasm  of  { 

(the  cells  takes  part  in  the  secretion  in  such  a  manner  that  it  becomes  itself  I 
a  constituent  of  the  secretion,  and  this  also  agrees  with  Heidexhaix's  7 ' 

1  >ee  Maly's  Jahresher.,  26.     See  also  Basch,  Ergebnisse  der  Physiologie,  2,  Abt.  I. 

2  In  regard  to  the  literature  on  the  action  of  various  foods  on  woman's  milk,  see 
Zalesky,  "Teller  die  Einwirkung  der  Xahrung  auf  die  Zusammensetzunc;  and  Xahr- 
haftigkeit  der  Frauenmileh,"  Berlin,  klin.  "Wochenschr. ,  1S.NN,  which  also  contains  the 
literature  on  the  importance  of  diet  on  the  composition  of  other  kinds  of  milk.  In 
regard  to  the  extensive  literature  on  the  influence  of  various  foods  on  the  milk  pro- 
duction of  animals,  see  Koarig,  Chem.  d.  menschl.  Xahrungs-  und  Genussmittel,  3.  Aufl., 
1,  298.     St-  al-o  Maly's  Jahresber.,  29,  30,  31. 

"Centralbl   f.  d.  med.  Wissensch. ,  1866,  337. 
*  Cited  from  Konig,  2,  235. 

Beck,  Maly's  Jahresber.,  85, 
8  Lehrbuch  d.  physiol.  und  pathol.  Chem.,  3.  Aufl.,  93. 
'Hermann's  Handbuch,  5,  Teil.  1,  3S0. 


458  MILK. 

views.  According  to  Basch's  *  researches  the  casein  is  formed  in  the 
mammary  gland  by  the  nucleic  acid  of  the  nucleus  being  set  free  and 
uniting  intraalveolar  with  the  transudated  serum,  thus  forming  a  nucleoalbu- 
min,  the  casein;  but  strong  objections  can  be  presented  to  such  a  view. 
^That  the  milk-fat  is  produced  by  a  formation  of  fat  in  the  protoplasm, 
/and  that  the  fat-globules  are  set  free  by  their  destruction,  is  a  generally 
admitted  opinion,  which,  however,  does  not  exclude  the  possibility  that 
the  fat  is  in  part  taken  up  by  the  glands  from  the  blood  and  eliminated 
with  its  secretion.  That  the  fats  of  the  food  can  pass  into  the  milk  follows 
from  the  investigations  of  Winternitz,  as  he  has  been  able  to  detect  the 
^  passage  of  iodized  fats  insthe  milk^/ Jantzen  has  shown  that  after  feeding 
iodized  casein,  the  milk-fat  of  goats  contained  a  little  iodine,  which  indicates 
that  the  iodized  milk-fat  could  also  have  a  different  origin.  Even  if  the 
casein  was  entirely  free  from  fat,  these  observations  do  not  seem  to  modify 
the  proof  of  the  investigations  of  Winternitz  and  others  (Capari, 
Paraschtschuk  2) .  The  abundant  quantities  of  iodized  fat  which  were 
eliminated  with  the  milk  in  the  last-mentioned  case  without  doubt  depend, 
at  least  in  great  part,  upon  the  iodized  fat  of  the  food ;  but  it  cannot  be 
said  that  all  of  the  milk-fat  containing  iodine  was  unchanged  iodized  fat 
of  the  food.  The  investigations  of  Spampani  and  Daddi,  Paraschtschuk 
and  others  on  the  passage  of  foreign  fats  into  the  milk  indicate  the  passage 
of  the  fat  of  the  food  into  the  milk,  although  we  are  still  uncertain  on  this 
point.  According  to  Soxhlet  the  fat  of  the  food  does  not  pass  into  the 
milk  directly,  but  is  destroyed  in  place  of  the  body-fat,  which  then  becomes 
available  and  is,  as  it  were,  pushed  into  the  milk.  Henriques  and  Han- 
sen 3  could  not  detect  any  mentionable  quantity  of  linseed-oil  in  the  milk 
after  feeding  with  this  oil;  the  milk-fat  was  not  normal,  but  had  a  higher 
iodine  equivalent  and  a  higher  melting-point,  from  which  they  also  concluded 
that  a  transformation  of  the  food-fat  in  the  glandular  cells  is  possible. 
/As  a  formation  of  fat  from  carbohydrates  in  the  animal  organism  is  at  the  \ 
'  present  day  considered  as  positively  proved,  it  is  likewise  possible  that 
the  milk-glands  also  produce  fats  from  the  carbohydrates  brought  to  them 
by  the  blood.  It  is  a  well-known  fact  that  an  animal  gives  off  for  a  long 
time,  daily,  considerably  more  fat  in  the  milk  than  it  receives  as  food,  and 
this  proves  that  at  least  a  part  of  the  fat  secreted  by  the  milk  is  produced 
from  proteids  or  carbohydrates,  or  perhaps  from  bothy  The  questions 
as  to  how  far  this  fat  is  produced  directly  in  the  milk-glands,  or  from  other 

1  Jahrbuch  f.  Kinderheilkunde,  1898. 

2  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  24;  Jantzen,  Centralbl.  f.  Physiol.,  15; 
Caspari,  Arch.  f.  (Anat.  u.)  Physiol.,  1899.  Supplbd. ;  Paraschtschuk,  Chem.  Cen- 
tralbl., 1903,  1. 

8  Spampani  and  Daddi,  Maly's  Jahresber.,  26;  Henriques  and  Hansen,  ibid.,  29. 
See  also  Basch,  Ergebnisse  d.  Physiol.,  2,  Abt.  I. 


CHEMISTRY  OF  MILK  SECRETION.  IV) 

organs  and  tissues,  and  brought  to  the  gland  by  means  01  the  blood,  can- 
not be  decided. 

The  origin  of  milk-sugar  is  not  known.  Muntz  calls  attention  to  the 
fact  that  a  number  of  very  widely  diffused  bodies  in  the  vegetable  king- 
dom— vegetable  mucilage,  gums,  pectin  bodies — yield  galactose  as  prod- 
ucts of  decomposition,  and  he  believes,  therefore,  that  milk-sugar  may 
be  formed  in  herbivora  by  a  synthesis  from  dextrose  and  galactose.  This 
origin  of  milk-sugar  does  not  apply  to  tarnivora,  as  they  produce  milk- 
sugar  when  fed  on  food  consisting  entirely  of  lean  meat.  The  observa- 
vations  of  Bert  and  Thierfelder1  that  a  mother-substance  of  the  milk- 
sugar,  a  saccharogen,  occurs  in  the  glands  cannot  give  further  explanation  as 
to  the  formation  of  milk-sugar,  as  the  nature  of  this  mother-substance  is  still 
unknown.  As  the  animal  body  has  undoubtedly  the  power  of  converting 
one  variety  of  sugar  into  another,  the  origin  of  the  milk-sugar  cannot  be 
sought  simply  in  the  dextrose  introduced  as  food  or  formed  in  the  body. 

The  passage  of  foreign  substances  into  the  milk  stands  in  close  connec- 
tion with  the  chemical  processes  of  milk-secretion. 

j  It  is  a  well-known  fact  that  milk  acquires  a  foreign  taste  from  the  food 
of  the  animal,  which  is  in  itself  a  proof  that  foreign  bodies  pass  into  the 
milk.  This  fact  becomes  of  special  importance  in  reference  to  such  injurious 
substances  as  may  be  introduced  into  the  organism  of  the  nursing  child  by 
means  of  the  milk. 

Among  these  substances  may  be  mentioned  opium  and  morphine,  which 
after  large  doses  pass  into  the  milk  and  act  on  the  child.  Alcohol  may  also 
pass  into  the  milk,  but  probably  not  in  such  quantities  as  to  have  any  direct 
action  on  the  nursing  child.2  Alcohol  is  claimed  to  have  been  detected  in 
the  milk  after  feeding  cows  with  brewer's  grains. 

Among  inorganic  bodies,  iodine,  arsenic,  bismuth,  antimony,  zinc,  lead, 
mercury,  and  iron  have  been  found  in  milk.  In  icterus  neither  bile-acids 
nor  bile-pigments  pass  into  the  milk.  J 

Under  diseased  condition^  no  constant  change  has  been  found  in  woman's 
milk.  In  isolated  cases  Schlossberger,  Joly  and  Filhol  3  have  observed 
indeed  a  markedly  abnormal  composition,  but  no  positive  conclusion  can  be 
derived  therefrom. 

The  changes  in  cow's  milk  in  disease  have  been  little  studied.  In  tuberculosis 
of  the  udder  Storch  4  found  tubercle  bacilli  in  the  milk,  and  he  i  lso  noted  that 
the  milk  became  more  and  more  diluted,  during  the  disease,  with  a  serous  liquid 
similar  to  blood-serum,  so  that  the  glands  finally,  instead  of  yielding  milk,  gave 

1  Muntz,  Compt.  rend.,  102;   Bert  and  Thierfelder,  foot-note  1,  page  437. 
'See  Klingemann,  Virchow's  Arch.,  126,  and  Rosemann,  Pfliiger's  Arch.,  7S. 

3  Schlossberger,  Annal.  d.  Chem.  u  Pharm.,  96;  Joly  and  Filhol,  cited  from  Gorup- 
Besanez,  Lehrb.,  4.  Aufl.,  438. 

4  See  Bang,  Cm  Tuberkulose  i  Koens  Y  ver  og  om  tuberkulos  Mfilk.  Xord.  med. 
Arkiv,  16,  and  also  Maly's  Jahresber.,  14,  170;  Storch,  Maly's  Jahresber.,  14. 


460  MILK. 

only  blood-serum  or  a  serous  fluid.  Husson  !  found  that  milk  from  murrain 
cows  contained  more  proteids  but  considerably  less  fat  and  (in  severe  cases)  less 
sugar  than  normal  milk. 

The  milk  may  be  blue  or  red  in  color,  due  to  the  development  of  micro-organisms. 

The  formation  of  concrements  in  the  exit-passages  of  the  cow's  udder  is  often 
observed.  They  consist  chiefly  of  calcium  carbonate,  or  of  carbonate  and  phos- 
phate with  only  a  small  amount  of  organic  substances. 

^ompt.  rend.,  73. 


CHAPTER  XV. 
URINE. 

Urine  is  the  most  important  excretion  of  the  animal  organism ;  it  is  the 
means  of  eliminating  the  nitrogenous  metabolic  products,  also  the  water  and 
the  soluble  mineral  substances;  and  in  many  cases  it  furnishes  important 
data  relative  to  the  metabolism,  quantitatively  by  its  variation,  and  quali- 
tatively by  the  appearance  of  foreign  bodies  in  the  excretion.  Moreover  in 
many  cases  we  are  able  from  the  chemical  or  morphological  constituents 
which  the  urine  abstracts  from  the  kidneys,  ureter,  bladder,  and  urethra 
to  judge  of  the  condition  of  these  organs;  and  lastly,  urinary  analysis 
affords  an  excellent  means  of  deciding  the  question  as  to  how  certain 
medicinal  agents  or  other  foreign  substances  introduced  into  the  organism 
are  absorbed  and  chemically  changed.  In  this  respect  especially  urinary 
analysis  has  furnished  very  important  particulars  in  regard  to  the  nature  of 
the  chemical  processes  taking  place  within  the  organism,  and  it  is  therefore 
not  only  an  important  aid  in  diagnosis  to  the  physician,  but  it  is  also  of 
the  greatest  importance  to  the  toxicologist  and  the  physiological  chemist. 

In  studying  the  secretions  and  excretions  the  relationship  must  be  sought 
between  the  chemical  structure  of  the  secreting  organ  and  the  chemical 
composition  of  its  secreted  products.  Investigations  with  respect  to  the 
kidneys  and  the  urine  have  led  to  very  few  results  from  this  standpoint. 
Although  the  anatomical  relation  of  the  kidneys  has  been  carefully  studied, 
their  chemical  composition  has  not  been  the  subject  of  thorough  analytical 
research.  In  cases  in  which  a  chemical  investigation  of  the  kidneys  has 
been  undertaken,  it  has  in  general  only  been  of  the  organ  as  such,  and  not 
of  the  different  anatomical  parts.  An  enumeration  of  the  chemical  con- 
stituents of  the  kidneys  known  at  the  present  time  can,  therefore,  have  only 
a  secondary  value. 

In  the  kidneys  we  find  proteids  of  different  kinds.  According 
to  Halliburton  the  kidneys  do  not  contain  any  albumin,  but  only  a 
globulin  and  a  nuclcoproteid.  The  globulin  coagulates  at  about  52°  C,  and 
the  nucleoproteid  contains  0.37  per  cent  phosphorus.  According  to 
L.  Liebermaxx  the  kidneys  contain  a  lecithalbumin,  and  he  ascribes  to 
this  body  a  special  importance  in  the  secretion  of  acid  urines.  The  kidneys 
also  contain,  according  to  Loxxberg,  a  mucin-like  substance.     This  sub- 

461 


462  URINE. 

stance  yields  no  reducing  body  on  boiling  with  acids  and  belongs  chiefly  to 
the  papillae,  and  is,  according  to  Lonnberg,  a  nucleoalbumin  (nucleopro- 
teid  ?).  The  cortical  substance  is  richer  in  another  nucleoalbumin  (nucleo- 
proteid)  unlike  mucin.  It  has  not  been  decided  what  relationship  this  last 
substance  bears  to  Halliburton  's  nucleoproteid.  According  to  Morner  1 
chondroitin-sulphuric  acid  occurs  as  traces. 

Fat  occurs  only  in  very  small  amounts  in  the  cells  of  the  tortuous  urinary 
passages.  Among  the  extractive  bodies  of  the  kidneys  one  finds  xanthine 
bodies,  also  urea,  uric  acid  (traces),  glycogen,  leucin,  inosite,  taurin,  and 
cystin  (in  ox-kidneys).  The  quantitative  analyses  of  the  kidneys  thus 
far  made  possess  little  interest.  Oidtmann  2  found  810.94  p.  m.  water, 
179.16  p.  m.  organic  and  0.99  p.  m.  inorganic  substance  in  the  kidney  of 
an  old  woman. 

The  fluid  collected  under  pathological  conditions,  as  in  hydronephrosis,  is  thin 
with  a  variable  but  generally  low  specific  gravity.  Usually  it  is  straw-yellow  or 
paler  in  color,  and  sometimes  colorless.  Most  frequently  it  is  clear,  or  only  faintly 
cloudy  from  white  blood-corpuscles  and  epithelium-cells;  in  a  few  cases  it  is  so 
rich  in  form-elements  that  it  appears  like  pus.  Proteid  occurs  generally  in 
small  amounts;  occasionally  it  is  entirely  absent,  but  in  a  few  rare  cases  the 
amount  is  nearly  as  large  as  in  the  blood-serum.  Urea  occurs  sometimes  in 
considerable  amounts  when  the  parenchyma  of  the  kidneys  is  only  in  part  atro- 
phied; in  complete  atrophy  the  urea  may  be  entirely  absent. 

I.  Physical  Properties  of  Urine. 

Consistency,  Transparency,  Odor,  and  Taste  of  Urine.  Under  physio- 
logical conditions  urine  is  a  thin  liquid  and  gives,  when  shaken  with  air,  a 
froth  which  quickly  subsides.  Human  urine  or  urine  from  carnivora,  which 
is  habitually  acid,  appears  clear  and  transparent,  often  faintly  fluorescent, 
immediately  after  voiding.  When  allowed  to  stand  for  a  little  while  human 
urine  shows  a  light  cloud  (nubecula),  which  consists  of  the  so  called  "mucus" 
and  generally  also  contains  a  few  epithelium- cells,  mucus-corpuscles,  and 
urate-granules.  The  presence  of  a  larger  quantity  of  urates  renders  the 
urine  cloudy,  and  a  clay-yellow,  yellowish-brown,  rose-colored,  or  often 
brick-red  precipitate  (sedimentum  lateritium)  settles  on  cooling,  because  of 
the  greater  insolubility  of  the  urates  at  the  ordinary  temperature  than  at 
the  temperature  of  the  body.  This  cloudiness  disappears  on  gently  warm- 
ing. In  new-born  infants  the  cloudiness  of  the  urine  during  the  first  4-5 
days  is  due  to  epithelium,  mucus-corpuscles,  uric  acid,  and  urates.  The 
urine  of  herbivora,  which  is  habitually  neutral  or  alkaline  in  reaction,  is 
very  cloudy  on  account  of  the  carbonates  of  the  alkaline  earths  present. 
Human  urine  may  sometimes  be  alkaline  under  physiological  conditions. 

1  Halliburton,  Journ.  of  Physiol.,  13,  Suppl.,  and  18;  Liebermann,  Pfluger's  Arch., 
50  and  54;  Lonnberg,  see  Maly's  Jahresber.,  20;  Morner,  Skand.  Arch.  f.  Physiol.,  6. 

2  Cited  from  v.  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  732. 


PHYSICAL  PROPERTIES  OF   THE   URINE.  463 

In  this  case  it  is  cloudy  due  to  the  earthy  phosphates,  and  this  cloudiness 
does  not  disappear  on  warming,  differing  in  this  respect  from  the  sedimen- 
turn  lateritium.  Urine  has  a  salty  and  faintly  bitter  taste  produced  by 
sodium  chloride  and  urea.  The  odor  of  urine  is  peculiarly  aromatic;  the 
bodies  which  produce  this  odor  are  unknown. 

The  color  of  urine  is  normally  pale  yellow  when  the  specific  gravity  is 
1.020.  The  color  otherwise  depends  on  the  concentration  of  the  urine  and 
varies  from  pale  straw-yellow,  when  the  urine  contains  small  amounts  of 
solids,  to  a  dark  reddish-yellow  or  reddish-brown  in  stronger  concentration. 
As  a  rule  the  intensity  of  the  color  corresponds  to  the  concentration,  but 
under  pathological  conditions  exceptions  occur  such  as  is  found  in  diabetic 
urine,  which  contains  a  large  amount  of  solids  and  has  a  high  specific 
gravity  and  a  pale-yellow  color. 

The  reaction  of  urine  depends  essentially  upon  the  composition  of  the 
food.  The  carnivora  void  an  acid,  the  herbivora,  as  a  rule,  a  neutral  or 
alkaline  urine.  If  a  carnivora  is  put  upon  a  vegetable  diet,  its  urine  may 
become  less  acid  or  neutral,  while  the  reverse  occurs  when  an  herbivora  is 
starved,  that  is,  when  it  lives  upon  its  own  flesh,  as  then  the  urine  voided  is 
acid. 

The  urine  of  a  healthy  man  on  a  mixed  diet  has  an  acid  reaction,  and 
the  sum  of  the  acid  equivalents  is  greater  than  the  sum  of  the  basic  equiva- 
lents. This  depends  upon  the  fact  that  in  the  physiological  combustion  of 
neutral  substances  (proteids  and  others)  within  the  organism  acids  arc  |  m  >- 
duced,  chiefly  sulphuric  acid,  but  also  phosphoric  and  organic  acids,  such  as 
hippuric,  uric,  and  oxalic  acid,  aromatic  oxyacids,  and  others.  From  this  it 
follows  that  the  acid  reaction  is  not  due  to  on  acid  alone.  The  ordinary 
view  that  the  acid  reaction  is  due  chiefly  to  dihydrogen  phosphates  is  therefore 
not  true.  The  various  acids  take  part  in  the  acid  reaction  in  proportion  to 
their  dissociation,  since,  according  to  the  ion  theory,  the  acid  reaction 
of  a  mixture  is  dependent  upon  the  number  of  hydrogen  ions  present. 

The  composition  of  the  food  is  not  the  only  influence  which  affects  the 
degree  of  acidity  of  human  urine.  For  example,  after  taking  food,  at  the 
beginning  of  digestion,  when  a  larger  amount  of  gastric  juice  containing 
hydrochloric  acid  is  secreted,  the  urine  may  be  neutral  or  even  alkaline.1 
The  statements  of  various  investigators  are  rather  contradictory  in  regard 
to  the  time  of  the  appearance  of  the  maximum  and  minimum  of  the  acid- 
ity, which  may  in  part  be  explained  by  the  different  individuality  and 
conditions  of  life  of  the  persons  investigated.  It  has  not  infrequently  been 
observed  that  perfectly  healthy  persons  in  the  morning  void  a  neutral  or 
alkaline  urine  which  is  cloudy  from  earthy  phosphates.  The  effect  of 
muscular  activity  on  the  acidity  of  urine  has  not  been  positively  determined. 


1  Contradictory  statements  are  found  in  Linossier,  Maly's  Jahresber.,  27. 


464  URINE. 

According  to  Hoffmann,  Ringstedt,  Oddi  and  Tarulli  muscular  work 
raises  the  degree  of  acidity,  but  Aducco  1  claims  hat  it  decreases  it.  Abun- 
dant perspiration  reduces  the  acidity  (Hoffmann)  . 

In  man  and  especially  in  carnivoia  it  seems  that  the  degree  of  acidity  of 
the  urine  cannot  be  increased  above  a  certain  point,  even  though  mineral 
acids  or  organic  acids  which  are  burnt  up  with  difficulty  are  ingested  in  large 
quantities.  When  the  supply  of  carbonates  of  the  fixzd  alkalies  stored  up 
in  the  organism  for  this  purpose  is  not  sufficient  to  combine  with  the  excess 
of  acid,  then  ammonia  is  split  off  from  the  proteids  o  their  decomposition 
products,  and  this  excess  of  acid  combines  therewith,  forming  ammonium 
salts,  which  pass  into  the  urine.  In  herbivora  such  a  combination  of  the 
excess  of  acid  with  ammonia  does  not  seem  to  take  place,  or  not  to  the  same 
extent,2  and  therefore  herbivora  soon  die  when  acids  are  given.  Neverthe- 
less the  degree  of  acidity  of  human  urine  may  be  easily  diminished  so  that 
the  reaction  becomes  neutral  or  alkaline.  This  occurs  after  the  taking  of 
carbonates  of  the  fixed  alkalies  or  of  such  alkali  salts  of  vegetable  acids — 
tartaric  acid,  citric  acid,  and  malic  acid — as  are  easily  burnt  into  carbonates 
in  the  organism.  Under  pathological  conditions,  as  in  the  absorption  of 
alkaline  transudates,  the  urine  may  become  alkaline. 

A  urine  with  an  alkaline  reaction  caused  by  fixed  alkalies  has  a  very 
different  diagnostic  value  from  one  whose  alkaline  reaction  is  caused  by 
the  presence  of  ammonium  carbonate.  In  the  latter  case  we  have  to  deal 
with  a  decomposition  of  the  urea  of  the  urine  by  the  action  of  micro-organ- 
isms. 

If  one  wishes  to  determine  whether  the  alkaline  reaction  of  the  urine  is 
due  to  ammonia  or  fixed  alkalies,  a  piece  of  red  litmus  paper  is  dipped  into 
the  urine  and  allow  it  to  dry  exposed  to  the  air  or  to  a  gentle  heat.  If  the 
alkaline  reaction  is  due  to  ammonia,  the  paper  becomes  red  again;  but  if 
it  is  caused  by  fixed  alkalies,  it  remains  blue. 

Determination  of  the  Acidity.  As  the  quantity  of  phosphoric  acid 
present  as  dihydrogen  salt,  as  above  stated,  cannot  be  used  as  a  measure 
of  the  acidity,  all  the  older  methods  suggested  for  the  estimation  of 
this  portion  of  the  phosphoric  acid  are  not  suited  for  acidity  deter- 
minations. We  now  determine  the  acidity  simply  by  acidimetric  methods 
by  titrating  with  N/10  caustic  alkali,  using  phenolphthalein  as  an  indicator 
(Naegeli,  Hober,  Folin).  On  account  of  the  color  of  the  urine  and  the 
presence  of  ammonium  salts  and  alkaline  earths,  this  method  cannot 
yield  entirely  exact  results.  The  greatest  error  depends  upon  the  alkaline 
earths,  which,  on  titration  with  caustic  alkali,  precipitate  as  earthy  phos- 
phates in  variable  amounts  and  of  variable  composition.      This  error  can 

1  Hoffmann,  see  Maly's  Jahresber.,  14;  Ringstedt,  ibid.,  20;  Oddi  and  Tarulli, 
ibid.,  24;    Aducco,  ibid.,  17. 

2  See  Winterberg,  Zeitschr.  f.  physiol.  Chem.,  25. 


DETERMINATION  OF  ACIDITY.  465 

be  prevented,  according  to  Folin,1  by  the  addition  of  neutral  potassium 
oxalate,  which  precipitates  the  lime,  and  in  this  way  the  disturbing  action 
of  the  ammonium  salts  Is  also  inhibited.  Perfectly  accurate  results  are 
not  obtained  by  this  method,  but  it  is  the  best  of  those  which  have  been 

suggested. 

It  is  performed  as  follows:  25  c.  c.  of  urine  are  placed  in  an  Erlen- 
inever  flask  (about  200  c.  c.  capacity),  treated  with  1-2  drops  of  £  percent 
phenolphthalein  solution,  and  shaken  with  15-20  grams  of  powdered  potas- 
sium oxalate  and  immediately  treated  with  N/10  caustic  soda  with  constant 
shaking  until  a  pronounced  pale-rose  color  appears. 

The  acidity,  as  determined  by  titration,  varies  considerably  under 
physiological  conditions,  but  calculated  as  hydrochloric  acid  it  amounts 
to  about  1.5-2.3  grams  in  man. 

By  titration  we  learn  the  amount  of  hydrogen  present  which  can  be 
substituted  by  a  metal,  i.e.,  the  acidity  in  the  ordinary  older  sense,  but  not 
the  true  acidity,  the  ion  acidity,  which  is  given  by  the  concentration  of  the 
hydrogen  ions  of  the  urine.  For  similar  reasons,  as  indicated  previously  in 
treating  of  the  alkalinity  of  the  blood-serum  (page  160),  the  ion  acidity 
cannot  be  determined  by  titration,  while  it  can  be  determined  according 
to  the  principle  of  the  electrometric  gas-chain  method  as  there  given.  Such 
estimations  have  been  made  by  v.  Rhorkr  and  by  Hober.2  For  normal 
urine  v.  Rhorer  found  as  a  minimum  410-7,  as  a  maximum  7610-7,  and  as 
an  average  3010-7.  Hober  found  4.710-7,  10010"7,  and  4910"7,  respectively. 
On  an  average  the  urine  contains  therefore  30-50  grams  of  hydrogen  ions 
in  10  million  liters,  and  as  in  the  same  quantity  of  purest  water  there  is 
contained  in  round  numbers  1  gram  of  hydrogen  ions,  the  urine  contains, 
therefore,  30-50  times  as  many  hydrogen  ions  as  the  water.  From  Hober 's 
investigations  it  also  follows  that  no  direct  relationship  exists  between  the 
titration  acidity  and  the  ion  acidity,  and  that  the  extent  of  these  two  acidi- 
ties may  be  independent  of  each  other. 

The  osmotic  pressure  of  the  urine  varies  considerably  even  under 
physiological  conditions.  The  limit  for  the  freezing-point  depression  has 
been  found  by  a  number  of  investigators  to  be  J  =  0.S7°  — 2.71°  C.3  After 
partaking  of  considerable  water  it  may  be  markedly  lower,  and  on  diminished 
supply  of  water  it  may  be  considerably  higher. 

According  to  Bugarsky  a  certain  relationship  exists  between  the  freezing- 
point  depression  and  the  specific  gravity,  namely  — -  =  constant  =  75.  This 
equation,  where  s  represents  the  specific  gravity,  has  no  general  application,  and 

1  Naegeli,  Zeitschr.  f.  physiol.  Chem.,  30;  Hober,  Hofmeister's  Beitrage,  3;  Folin, 
Amer.  Journ.  of  Physiol.,  9. 

2  v.  Rhorer,  Pfliiger's  Arch.,  86;   Hober,  1.  c. 
'  See  Strauss,  Zeitschr.  f.  klin.  Med..  4". 


466  URINE. 

according  to  Stetrer,1  is  only  approximate  for  normal  urines.  The  validity 
of  the  relationship,  as  found  by  Bugarsky,  between  the  electric  conductivity 
and  the  ash  content  of  the  urine,  seems  also  to  require  further  proof. 

The  specific  gravity  of  urine,  which  is  dependent  upon  the  relationship 
exis.ing  between  the  quantity  of  water  secreted  and  the  solid  urinary  con- 
stituents, especially  the  u  ea  and  sodium  chloride,  may  vary  onsiderably, 
but  is  generally  1.017-1.020.  After  drinking  large  quantities  of  water  it 
may  fall  to  1.002,  while  after  profuse  perspiration  or  after  drinking  very 
little  water  it  may  rise  to  1.035-1.040.  In  new-born  infants  the  specific 
gravity  is  low,  1.007-1.005.  The  determination  of  the  specific  gravity  is 
an  important  means  of  learning  the  average  amount  of  solids  eliminated 
from  the  organism  with  the  urine,  and  on  this  account  the  determination 
becomes  of  true  value  only  when  at  the  same  time  the  quantity  of  urine 
voided  in  a  given  time  is  determined.  The  different  portions  of  urine 
voided  in  the  course  of  the  twenty-four  hours  are  collected,  mixed  together, 
the  total  quantity  measured,  and  then  the  specific  gravity  taken. 

The  determination  of  the  specific  gravity  is  most  accurately  obtained  with 
the  pyknometer.  For  ordinary  cases  the  specific  gravity  may  be  deter- 
mined with  sufficient  accuracy  by  means  of  areometers.  .  The  areometers 
found  in  the  trade,  or  urinometers,  are  graduated  from  1.000  to  1.040;  for 
exact  observations  it  is  better  to  use  two  urinometers,  one  graduated  from 
1.000  to  1.020,  and  the  other  from  1.020  to  1.040. 

To  determine  the  specific  gravity  of  urine,  if  necessary  filter  the  urine, 
or  if  it  contains  a  urate  sediment,  first  dissolve  it  by  gentle  heat,  then  pour 
the  clear  urine  into  a  dry  cylinder,  avoiding  the  formati  n  of  froth.  Air- 
bubbles  or  froth,  when  present,  must  be  removed  with  a  glass  rod  or  filter- 
paper.  The  cylinder,  which  should  be  about  four  fifths  full,  must  be  vide 
enough  to  allow  the  urinometer  to  swim  freely  in  the  liquid  without  touch- 
ing the  sides.  The  cylinder  and  urinometer  should  both  be  dry  or  previously 
washed  with  the  urine.  On  reading,  the  eye  is  brought  on  a  level  with  the 
lower  meniscus — which  occurs  when  the  surface  of  the  liquid  and  the  lower 
limb  of  the  meniscus  coincide;  the  reading  is  then  made  from  the  point 
where  this  curved  line  cuts  the  scale  of  the  urinometer.  If  the  eye  is  not 
in  the  same  horizontal  plane  with  the  convex  line  of  the  meniscus,  but  is 
too  high  or  too  low,  the  surface  of  the  liquid  assumes  the  shape  of  an  ellipse, 
and  the  reading  in  this  position  is  incorrect.  Before  reading  press  the 
urinometer  gently  down  into  the  liquid  and  then  allow  it  to  rise,  and  wait 
until  it  is  at  rest. 

Each  urinometer  is  graduated  for  a  certain  temperature,  which  is  marked 
on  the  instrument,  or  at  least  on  the  best.  If  the  urine  is  not  at  the  proper 
temperature,  the  following  corrections  must  be  made:  For  every  three 
degrees  above  the  normal  temperature  one  unit  of  the  last  order  is  added  1o 
the  reading,  and  for  every  three  degrees  below  the  normal  temperature  one 
unit  (as  above)  is  subtracted  from  the  specific  gravity  observed.  For  exam- 
ple, when    a    urinometer   graduated    for  15°  C.  shows   a  specific  gravity 

'Bugarsky,  Pfliiger's  Arch..  fiS;    Steyrer,  Hofmeister's  Beitrage,  2. 


UREA.  407 

of    1.017    at  24°  C.,  then  the  specific    gravity  at  15°  C.  =  1.017 +  0.003  = 
1.020. 

When  great  exactitude  is  required,  as,  for  instance,  a  determination  to 
the  fourth  decimal  point,  we  make  use  of  a  urinometer  constructed  by 
LoHN  STEIN.1  JOLLES  :>  has  also  devised  a  small  urinometer  for  the  deter- 
mination of  the  specific  gravity  of  small  amounts  of  urine,  20-25  c.  c.  The 
specific  gravity  may  also  be  di  termined  by  the  Westphal  hydrostatic 
balance. 

II.  Organic,   Physiological  Constituents  of  the  Urine. 

+  XH 

Urea,  Ur,  CON\H4  =  CO<;-tt2.  has  been  synthetically  prepared  in  sev- 
eral ways,  especially,  as  Wohler  showed  in  1828,  by  the  metameric  trans- 
formation of  ammonium  isocyanate:  CO.N.XH4  =  CO(NH,)2.  It  is  also 
produced  by  the  decomposition  or  oxidation  of  certain  bodies  found  in  the 
animal  organisms,  such  as  purin  bodies,  creatine,  arginin,  etc. 

Urea  Ls  found  most  abundantly  in  the  urine  of  carnivora  and  man,  but  in 
smalle"  quantities  in  that  of  herbivora.  The  quantity  in  human  urine  is 
ordinarily  20-30  p.  m.  It  has  also  been  found  in  small  quantities  in  the 
urine  of  amphibians,  fishes,  and  certain  birds.  Urea  occurs  in  the  perspira- 
tion in  small  quantities,  and  a>  traces  in  th  blood  and  in  most  of  the  animal 
fluids.  It  also  occurs  in  rather  large  quantities  in  the  blood,  liver,  muscle,3 
an  I  bile  4  of  sharks.  Urea  is  also  found  in  certain  tissue  and  organs  of 
mammals,  especially  in  the  liver  and  spleen,  although  only  in  small  amounts. 
Under  pathological  conditions,  as  in  obstructed  excretion,  urea  may  appear 
to  a  considerable  extent  in  the  animal  fluids  and  tissues. 

The  quantity  of  urea  which  is  voided  in  twenty-four  hours  on  a  mixed 
diet  is  in  a  grown  man  about  30  grams,  in  women  somewhat  less.  While 
children  void  less,  the  excretion  relative  to  their  body-weight  is  greater  than 
in  grown  persons.  The  physiological  significance  of  urea  lies  in  the  fact 
that  this  body  forms  in  man  and  carnivora,  from  a  quantitative  standpoint, 
the  most  important  nitrogenous  end  product  of  the  metabolism  of  proteid 
bodies.  On  this  account  the  elimination  of  urea  varies  to  a  great  extent 
With  the  katabolism  of  the  proteid,  and  above  all  with  the  quantity  of 
absorbable  proteids  in  the  food  ingested.  The  elimination  of  urea  is  greatest 
after  an  exclusive  meat  diet,  and  lowest,  indeed  less  than  during  starvation 
after  the  consumption  of  non-nitrogenous  substances,  since  these  diminish 
the  metabolism  of  the  proteids  of  the  body. 

If  the  consumption  of  the  proteids  of  the  body  is  increased,  then  the 


1  Pfliiger's  Arch..  59:  Chem.  Centralbl.,  1895,  1,  and  1896,  2. 

7  Wien.  med.  Presse,  1897,  No.  8. 

sv.  Schroder,  Zeitschr.  f.  physiol.  Chem.,  14. 

*  Hammaratea,  ibid.,  24. 


468  URINE. 

elimination  of  nitrogen  is  correspondingly  increased.  This  is  found  to  be 
the  case  in  fevers,  cachexia,  diabetes,  after  poisoning  with  arsenic,  antimony, 
phosphorus,  and  other  protoplasmic  poisons,  by  a  diminished  supply  of  oxygen 
— as  in  severe  and  continuous  dyspnoea,  poisoning  with  carbon  monoxide, 
hemorrhage,  etc.  In  these  cases  it  used  to  be  considered  that  the  rise  in  the 
excretion  of  nitrogen  was  due  to  an  increased  elimination  of  urea,  because  no 
exact  difference  was  made  between  the  quantity  of  urea  and  of  total  nitrogen 
in  the  urine.  Recent  researches  have  conclusively  demonstrated  the  un trust- 
worthiness of  these  observations.  Since  Pfluger  and  Bohland  have 
shown  that  16  per  cent  of  the  total  nitrogen  of  the  urine  exists  under  physio- 
logical conditions  as  other  combinations,  not  urea,  attention  has  been  called 
to  the  relationship  of  the  different  nitrogenous  constituents  of  the  urine  to 
each  other,  and  it  has  been  found,  under  pathological  conditions,  that  this 
relationship  may  vary  considerably,  especially  in  regard  to  the  urea. 
We  have  numerous  determinations  by  different  investigators,  such  as  Boh- 
land, E.  Schultze,  Camerer,  Voges,  Morner  and  Sjoqvist,  Gumlich, 
Bodtker,1  and  others,  on  the  relationship  of  the  different  nitrogenous  con- 
stituents to  each  other  in  the  normal  urine  of  adults.  Sjoqvist  has  made 
similar  determinations  on  new-born  babes  from  1-7  days  old.  From  all 
these  analyses  we  obtain  the  following  figures  (A  for  adults  and  B  for  new- 
born babes).     Of  the  total  nitrogen  there  exists: 

a.  b. 

Per  Cent.  Per  Cent. 

Urea 84-91  73-76 

Ammonia 2-5  7.8-9.6 

Uric  acid 1-3  3.0-8.5 

Remaining  nitrogenous  substances  (extractives)  ...  .     7-12  7.3-14.7 

The  different  relationship  between  uric  acid,  ammonia,  and  urea  nitrogen 
in  children  and  adults  is  remarkable,  since  the  urine  of  children  is  consider- 
ably richer  in  uric  acid  and  ammonia,  and  considerably  poorer  in  urea,  than 
the  urine  of  adults.  The  absolute  quantity  of  urea  nitrogen  in  adults 
amounts  to  about  10-16  grams  per  day.  In  disease  the  proportion  of  the 
nitrogenous  substances  may  be  markedly  changed,  and  a  decrease  in  the 
quantity  of  urea  and  an  increase  in  the  quantity  of  ammonia  have  been 
observed  in  certain  diseases  of  the  liver.  This  will  be  considered  in  detail  in 
connection  with  the  formation  of  urea  in  the  liver.  It  is  natural  that  there 
should  be  a  diminished  formation  of  urea  after  a  decrease  in  the  inges- 
tion of  proteids  or  in  a  lowered  katabolism.     In  diseases  of  the  kidneys 

^fliiger  and  Bohland,  Pfli'iger's  Arch.,  38  and  43;  Bohland,  ibid.,  43;  Schultze, 
ibid.,  4.">;  Camerer,  Zeitschr.  f.  Biologie,  24,  27,  and  28;  Voges,  Ueber  die  Mischung 
der  stickstoffhaltigen  Bestandtheile  im  Harn,  etc.  (Inaug.-Diss.,  Berlin,  1892),  cited 
from  Maly's  Jahresber.,  22;  K  Morner  and  Sjoqvist,  Skand.  Arch.  f.  Physiol.,  2. 
See  also  Sjoqvist,  Nord.  med.  Arkiv.,  1892,  No.  36,  and  1894,  No.  10;  Gumlich,  Zeitschr. 
f.  physiol.  Chem.,  17;  Bodtker,  see  .Maly's  Jahresber.,  2"». 


FORMATION  OF   UREA.  469 

"which  disturb  or  destroy  the  integrity  of  the  epithelium  of  the  convoluted 
urinary  passages  the  elimination  of  urea  is  considerably  diminished. 

Recently  by  means  of  Pfaundler's  method,  by  precipitating  the  urine  with 
phosphotungstic  acid  and  closely  studying  the  precipitate  as  well  as  the  filtrate, 
it  has  been  possible  to  learn  further  about  the  division  of  the  nitrogen  of  the  urine 
and  also  to  determine  the  amount  of  amino-acid  nitrogen.  Pfaundler  1  found 
in  one  case  4.76  per  cent  of  the  total  nitrogen  as  so-called  amino-acid  nitrogen 
and  Kri'ger  and  Schmid  0-6  per  cent.  According  to  v.  Jakscii,2  the  amino- 
acid  nitrogen  is  increased  in  diseases  of  the  liver,  typhoid,  and  diabetes.  In 
normal  human  urines  the  amino-acid  nitrogen  must  not  be  above  1.5-3  per  cent 
of  the  total  nitrogen  (v.  Jaksch).  5.16  to  8.5  per  cent  of  the  total  nitrogen  comes 
from  the  ammonia  and  purin  bodies.  The  methods  have  not  been  sufficiently 
developed  and  tested  in  order  to  allow  of  positive  conclusions.  Those  bodies 
which  arc  represented  by  the  amino-acid  nitrogen  are  only  known  to  a  slight 
degree  (hippuric  acid,  oxyproteic  acid,  and  others). 

Formation  of  Urea  in  the  Organism.  The  experiments  to  produce  urea 
directly  from  proteids  by  oxidation  have  led  to  the  formation  of  some  guan- 
idine,  but  urea  has  not  been  obtained  positively.  On  the  hydrolysis  of  pro- 
teids arginin  has  been  found  among  other  products,  and  as  it  is  also  produced 
in  tryptic  digestion,  it  is  possible  that  a  small  portion  of  the  urea  is 
produced  in  this  manner,  according  to  the  kind  of  proteid  (Drechsel, 
Kossel,  see  Chapter  II).  Drechsel  claims  that  about  10  per  cent  of  the 
urea  can  be  accounted  for  in  this  way.  A  part  of  the  urea  may  be  produced 
by  the  action  of  alkalies  on  creatine  or  creatinine,  btit  this  is  hardly  probable. 

The  amino  acids  are  also  considered  as  mother-substances  of  urea.  By 
the  researches  of  Schultzen  and  Nencki  and  Salkowski  with  leucin  and 
glycocoll  and  those  of  v.  Knieriem  with  asparagin,  it  has  been  shown  that 
the  amino  acids  are  in  part  converted  into  urea  in  the  animal  organism. 
The  investigations  by  Salaskin  with  the  three  amino  acids,  glycocoll, 
leucin,  and  aspartic  acid,  have  unmistakably  shown  that  the  living  dog- 
liver,  supplied  with  arterial  blood,  has  the  property  of  transforming  the 
above  amino  acids  into  urea  or  a  closely  allied  substance.  The  researches 
of  Lobwi  with  the  "urea-forming"  enzyme  of  the  liver,  discovered  by 
Richtet,  and  glycocoll  or  leucin,  as  also  the  researches  of  Ascoli,3  have 
led  to  similar  results,  but  it  must  be  remarked  that  we  have  no  proof 
as  to  the  identity  of  the  newly  formed  substance  with  urea.  Nothing  can 
be  stated  in  regard  to  the  extent  of  formation  of  amino  acids  in  the  physio- 
logical destruction  of  proteids  in  the  animal  body,  with  the  exception  of 
those  formed  in  the  intestinal  digestion.  The  possibility  of  such  a  forma- 
tion of  urea  is  beyond  dispute. 

1  Pfaundler,  Zeitschr.  f.  physiol.  Chem.,  30;  Kriiger  and  Schmid,  ibid.,  31. 

2  v.  Jaksch,  Zeitschr.  f.  klin.  Med.,  50. 

3  Schultzen  and  Nencki,  Zeitschr.  f.  Biologie,  8;  v.  Knieriem,  ibid.,  10;  Salkowski, 
Zeitschr.  f.  physiol.  Chem.,  4;  Salaskin,  ibid.,  25;  Loewi,  ibid.,  25;  Richtet,  Compt. 
*end.,  118,  and  Compt.  rend.  soc.  biol.,  49;  Ascoli,  Pfluger's  Arch.,  72. 


470  URINE. 

Nothing  positive  can  be  said  in  regard  to  the  manner  in  which  this 
formation  of  urea  occurs;  but  it  is  admitted  that  it  is  partly  a  formation 
from  ammonia  and  partly  from  carbamic  acid. 

The  possibility  of  a  formation  of  urea  from  ammonia  has  been  positively 
shown.  Thus  the  researches  of  v.  Knieriem,  Salkowski,  Feder,  I.  Munk, 
Coranda,  Schmiedeberg  and  Fr.  Walter,  and  Hallerworden,  Pohl 
and  Munzer,1  on  the  behavior  of  ammonium  salts  in  the  animal  body  and 
the  elimination  of  the  ammonia  under  various  conditions,  have  shown 
that  not  only  ammonium  carbonate,  but  also  such  ammonium  salts  which 
are  burnt  into  carbonate  in  the  organism,  are  transformed  into  urea  by 
carnivora  as  well  as  herbivora.  v.  Schroder,2  by  irrigating  the  living 
dog's  liver  with  blood  treated  with  ammonium  carbonate  or  ammonium 
formate,  has  shown  that  the  formation  of  urea  takes  place,  at  least  in  part, 
in  this  organ.  Nencki,  Pawlow  and  Zaleski  and  Salaskin  3  have  found 
that  in  dogs  the  quantity  of  ammonia  in  the  blood  from  the  portal  vein  is 
considerably  greater  than  that  from  the  hepatic  vein,  and  they  claim  that 
the  liver  retains  in  great  part  the  ammonia  thus  supplied.  The  formation 
of  urea  from  ammonia  in  the  liver  is  a  positively  proved  fact,  and  the  urea 
formation  from  ammonium  carbonate  is  to  be  considered  as  a  synthesis 
with  the  elimination  of  water. 

We  have  also  important  observations  which  give  support  to  the  views  of 
Schultzen  and  Nencki,4  namely,  that  the  amino  acids  are  transformed 
into  urea  with  carbamic  acid  as  an  intermediate  step.  Drechsel  has 
shown  that  the  amino  acids  yield  carbamic  acids  by  oxidation  in  alkaline 
fluid  outside  of  the  organism,  and  he  obtained  urea  from  ammonium  car- 
bamate by  passing  an  alternating  electric  current  through  its  solution, 
i.e.,  by  alternate  oxidation  and  reduction.  Drechsel  has  also  been 
able  to  detect  small  quantities  of  carbamates  in  blood,  and  later  in  con- 
junction with  Abel  he  detected  carbamic  acid  in  alkaline  horse's  urine. 
Drechsel  therefore  accepts  the  formation  of  urea  from  ammonium  car- 
bamate, and  according  to  him  the  alternating  oxidation  and  reduction  take 
place  in  the  following  way: 

H4N.O.CO.NH2+0  =  H2N.O.CO.NH2+H20 

Ammonium  carbamate 

and 

H2N.O.CO.NH2+H2=H2N.CO.NH2+H20. 

Urea 

1  v.  Knieriem,  Zeitschr.  f.  Biologie,  10;  Feder,  ibid.,  13;  Salkowski,  Zeitschr.  f. 
Biologie,  1;  Munk,  ibid.,  2;  Coranda,  Arch.  f.  exp.  Path.  u.  Pharm.,  12;  Schmiede- 
berg and  Walter,  ibid.,  7;  Hallerworden,  ibid.,  10;  Pohl  and  Munzer,  Arch.  f.  exp. 
Path.  u.  Pharm.,  43. 

2  Arch.  f.  exp.  Path.  u.  Pharm.,  15.     See  also  Salomon,  Virchow's  Arch.,  97. 

8  Arch,  des  sciences  biol.  de  St.  P6tersbourg,  4;  see  also  Chapter  VI,  page  202. 
*  Zeitschr.  f.  Biologie,  8. 


FORMATION  OF   UREA.  471 

Abel  and  Muirhead1  have  later  observed  an  abundant  elimination  of 
carbamic  acid  in  human  and  dog's  urine  after  the  administration  of  large 
quantities  of  milk  of  lime,  and  the  probability  of  the  regular  appearance  of 
this  acid  in  normal  acid  human  and  dog's  urine  has  been  demonstrated  by 
M.  Nencki  and  Hahn.2  These  last-mentioned  investigators  have  also 
given  very  important  support  to  the  theory  of  the  formation  of  urea  from 
ammonium  carbamate  by  observations  on  dogs  with  Eck's  fistula.  In  this 
case  the  portal  vein  is  directly  connected  with  the  inferior  vena  cava,  and 
a  communication  is  thus  established  so  that  the  blood  of  the  portal  vein 
flows  directly  into  the  vena  cava,  without  passing  through  the  liver. 
Nencki  and  Hahn  observed  violent  symptoms  of  poisoning  in  dogs  oper- 
ated upon  by  Pawlow  and  Massen,  and  these  symptoms  were  quite  identi- 
cal with  those  obtained  on  introducing  carbamate  into  the  blood.  These 
symptoms  also  appear  after  the  introduction  of  carbamate  into  the  stomach, 
while  the  introduction  of  carbamate  into  the  stomach  of  a  normal  dog  had 
no  action.  As  these  observers  also  found  that  the  urine  of  the  dog  on  which 
the  operation  was  made  was  richer  in  carbamate  than  that  of  the  normal 
dog,  they  concluded  that  the  symptoms  were  due  to  the  non-transformation 
of  the  ammonium  carbamate  into  urea  in  the  liver,  and  they  consider  the 
ammonium  carbamate  as  the  substance  from  which  the  urea  is  derived  in 
the  liver  of  mammals. 

The  view  as  to  the  formation  of  urea  from  ammonium  carbamate  does 
not  contradict  the  above  statement  as  to  the  transformation  of  carbonates 
into  urea,  since  we  can  imagine  that  the  carbonate  is  first  converted  into 
carbamate  with  the  expulsion  of  a  molecule  of  water,  and  that  this  then  is 
transformed  into  urea  with  the  expulsion  of  a  second  molecule  of  water. 

F.  Hofmeister  3  has  found  in  the  oxidation  of  different  members  of  the 
fatty  series,  as  well  as  in  amino  acids  and  proteids,  that  urea  was  formed 
in  the  presence  of  ammonia,  and  he  therefore  suggests  the  possibility  that 
urea  may  be  formed  by  an  oxidation-synthesis.  According  to  him,  in  the 
oxidation  of  nitrogenous  substances  a  radical  CONH2,  containing  the 
amid  group,  unites  at  the  moment  of  formation  with  the  radical  NH2 
remaining  on  the  oxidation  of  ammonia,  forming  urea. 

Besides  the  above-mentioned  theories  as  to  the  formation  of  urea,  there 
are  others  which  will  not  be  given,  because  the  only  theory  which  has  thus 
far  been  positively  demonstrated  is  the  formation  of  urea  from  ammonium 
compounds  and  amino  acids  in  the  liver. 

1  Drechsel,  Ber.  d.  sachs.  Gesellsch.  d.  Wissensch.,  1875.  See  also  Journ.  f.  prakt. 
Chem.  (N.  P.),  12,  16,  and  22;  Abel,  Du  Bois-Reymond's  Arch.,  1S91;  Abel  and 
Muirhead,  Arch.  f.  exp.  Path.  u.  Pharm.,  31. 

2  Hahn,  Massen,  Nencki  et  Pawlow,  La  fistule  d'Eck  de  la  veine  cave  inferieur  et 
de  la  veine  porte,  etc.     Arch,  des  sciences  biol.  de  St.  P6tersbourg,  1,  No.  4,  1892. 

'Arch.  f.  exp.  Path.  u.  Pharm.,  37. 


472  URINE. 

The  liver  is  the  only  organ  in  which,  up  to  the  present  time,  a  formation 
of  urea  has  been  directly  detected; x  and  the  question  arises,  what  importance 
has  this  urea  formation  which  takes  place  in  the  liver?  Is  the  urea  wholly 
or  chiefly  formed  in  the  liver? 

If  the  liver  is  the  only  organ  capable  of  forming  urea,  it  is  to  be  expected, 
on  the  extirpation  or  atrophy  of  that  organ,  that  a  reduced  or,  in  short  experi- 
ments, at  least  a  strongly  diminished  elimination  of  urea  should  occur.  As 
at  least  a  part  of  the  urea  is  formed  in  the  liver  from  ammonium  compounds  r 
a  simultaneous  increase  in  the  elimination  of  ammonia  is  to  be  expected. 

The  extirpation  and  atrophy  experiments  on  animals  made  by  different 
methods  by  Nencki  and  Hahn,  Slosse,  Lieblein,  Nencki  and  Pawlow, 
Salaskin  and  Zaleski  2  have  shown  that  a  rather  marked  increase  of 
ammonia  and  a  diminished  elimination  of  urea  take  place  after  the  opera- 
tion, but  also  that  there  are  cases  in  which,  irrespective  of  the  pronounced 
atrophy,  an  abundant  formation  of  urea  occurs,  and  no  appreciable,  if  any, 
change  in  the  proportion  of  ammonia  to  the  total  nitrogen  and  urea  is 
observed.  After  shutting  out  the  organs  of  the  posterior  part  of  the 
body,  especially  the  liver  and  kidneys,  from  the  circulation,  Kaupmann  3  also 
found  an  important  increase  in  the  urea  of  the  blood,  and  these  different 
observations  show  that  the  liver  is  not  the  only  organ,  in  the  various  animals 
experimented  upon,  in  which  urea  is  formed. 

The  observations  made  by  numerous  investigators  4  on  human  beings 
with  cirrhosis  of  the  liver,  acute  yellow  atrophy,  and  phosphorus  poisoning 
have  led  to  the  same  result.  These  investigations  teach  that  in  certain 
cases  the  proportion  of  the  nitrogenous  substances  may  be  so  changed 
that  urea  is  only  50-60  per  cent  of  the  total  nitrogen,  while  in  other  cases, 
on  the  contrary,  even  in  very  extensive  atrophy  of  the  liver-cells,  the  forma- 
tion of  urea  is  not  diminished,  neither  is  the  proportion  between  the  total 
nitrogen,  urea,  and  ammonia  essentially  changed.  Even  in  the  cases  in 
which  the  formation  of  urea  was  relatively  diminished  and  the  elimination 

1  In  regard  to  the  investigations  of  Prevost  and  Dumas,  Meissner,  Voit,  Gr<§hant, 
Gscheidlen  and  Salkowski,  and  others,  on  the  role  of  the  kidneys  in  the  formation  of 
urea,  see  v.  Schroeder,  Arch.  f.  exp.  Path.  u.  Pharm.,  15  and  19,  and  Voit,  Zeitschr. 
f.  Biologie,  4. 

2  Nencki  and  Hahn,  I.e.;  Slosse,  Du  Bois-Reymond 's  Arch.,  1890;  Lieblein,  Arch, 
f.  exp.  Path.  u.  Pharm.,  33;  Nencki  and  Pawlow,  Arch,  des  scienc.  de  St.  P6tersbourg, 
5.  See  also  v.  Meister,  Maly's  Jahresber.,  25;  Salaskin  and  Zaleski,  Zeitschr.  f. 
physiol.  Chem.,  29. 

3  Compt.  rend.  Soc.  biol.,  40,  and  Arch,  de  Physiol.  (5),  6. 

4  See  Hallerworden,  Arch.  f.  exp.  Path.  u.  Pharm.,  12;  Weintraud,  ibid.,  31;  Miinzer 
and  Winterberg,  ibid.,  33;  Stadelmann,  Deutsch.  Arch.  f.  klin.  Med.,  33;  Fawitzki,. 
ibid.,  45;  Miinzer,  ibid.,  52;  Frankel,  Berlin  klin.  Wochenschr.,  1878;  Richter,  ibid.r 
1890;  Morner  and  Sjoqvist,  Skand.  Arch.  f.  Physiol.,  2,  and  Sjoqvist,  Nord.  Med. 
Arkiv.,  1892;  Gumlich,  Zeitschr.  f.  physiol.  Chem.,  17;  v.  Noorden,  Lehrb.  d.  Pathol, 
des  Stoffwechsels,  287. 


PROPERTIES  OF   UREA.  473 

of  ammonia  considerably  increased  further  investigation  must  be  instituted 
before  it  will  be  possible  to  assume  a  reduced  ability  of  the  organism  to 
produce  urea.  An  increased  elimination  of  ammonia  may,  as  shown  by 
MiJNZER  in  the  case  of  acute  phosphorus  poisoning,  be  dependent  upon 
the  formation  of  abnormally  large  quantities  of  acids,  caused  by  abnormal 
metabolism,  and  these  acids  require  a  greater  quantity  of  ammonia  for  their 
neutralization  according  to  the  law  of  the  elimination  of  ammonia,  which 
will  be  given  later.  That  an  abnormal  formation  of  acid  occur.-  after  the 
cutting  out  of  the  liver  has  been  especially  shown  by  Salaskix  and 
Zalesei.1 

For  the  present  we  are  not  justified  in  the  statement  that  the  liver  is 
the  only  organ  in  which  urea  is  formed,  and  continued  investigation  only 
can  yield  further  information  as  to  the  extent  and  importance  of  the  forma- 
tion of  urea  from  ammonium  compounds  in  the  liver. 

Properties  and  Reactions  of  Urea.  Urea  crystallizes  in  needles  or  in 
long,  colorless,  four-sided,  often  hollow,  anhydrous  rhombic  prisms.  It  has 
a  neutral  reaction  and  produces  a  cooling  sensation  on  the  tongue  like  salt- 
peter. It  melts  at  132°  C.  At  ordinary  temperatures  it  dissolves  in  an  equal 
weight  of  water  and  in  five  parts  alcohol;  it  requires  one  part  boiling  alcohol 
for  solution;  it  is  insoluble  in  alcohol-free  ether,  and  also  in  chloroform. 
If  urea  in  substance  is  heated  in  a  test-tube,  it  melts,  decomposes,  gives  off 
ammonia,  and  leaves  finally  a  non-transparent  white  residue  which,  among 
other  substances,  contains  cyanuric  acid  and  biuret,  which  latter  dissolves 
in  water,  giving  a  beautiful  reddish-violet  liquid  with  copper  sulphate  and 
alkali  (biuret  reaction).  On  heating  with  baryta-water  or  caustic  alkali,  also 
in  the  so-called  alkaline  fermentation  of  urine  caused  by  micro-organisms, 
urea  splits  into  carbon  dioxide  and  ammonia  with  the  addition  of  water. 
The  same  decomposition  products  are  produced  when  urea  is  heated  with 
concentrated  sulphuric  acid.  An  alkaline  solution  of  sodium  hypobromite 
decomposes  urea  into  nitrogen,  carbon  dioxide,  and  water  according  to  the 
equation 

CON2H4+  3XaOBr  =  3NaBr+  C02+  2H20+  X2. 

With  a  concentrated  solution  of  furfurol  and  hydrochloric  acid  urea 
in  substance  gives  a  coloration  passing  from  yellow,  green,  blue,  to  violet, 
and  then  beautiful  purple- violet  after  a  few  minutes  (Schiff's  reaction). 
According  to  Huppert  2  the  test  is  best  performed  by  taking  2  c.  c.  of  a 
concentrated  furfurol  solution,  4-6  drops  of  concentrated  hydrochloric  acid, 
and  adding  to  this  mixture,  which  must  not  be  red,  a  small  crystal  of  urea. 
A  deep  violet  coloration  appears  in  a  few  minutes. 

Urea  forms  crystalline  combinations  with  many  acids.     Among  these 

1  Zeitschr.  f.  physiol.  Chem.,  29. 

1  Huppert-N'eubauer,  Analyse  des  Harnes,  10.  Aufl.,  296. 


474  URINE. 

the    one  with  nitric  acid  and  the  one  with  oxalic  acid  are  the  most 
important. 

Urea  Nitrate,  CO(NH2)2.HN03.  On  crystallizing  quickly  this  com- 
bination forms  thin  rhombic  or  six-sided  overlapping  tiles,  or  colorless 
plates,  with  an  angle  of  82°.  When  crystallizing  slowly,  larger  and 
thicker  rhombic  pillars  or  plates  are  obtained.  This  combination  is  rather 
easily  soluble  in  pure  water,  but  is  considerably  less  soluble  in  water  con- 
taining nitric  acid;  it  may  be  obtained  by  treating  a  concentrated  solution 
of  urea  with  an  excess  of  strong  nitric  acid  free  from  nitrous  acid.  On 
heating  this  combination  it  volatilizes  without  leaving  a  residue. 

This  compound  may  be  employed  with  advantage  in  detecting  small  amounts 
of  urea.  A  drop  of  the  concentrated  solution  is  placed  on  a  microscope-slide  and 
the  cover-glass  placed  upon  it;  a  drop  of  nitric  acid  is  then  placed  on  the  side 
of  the  cover-glass  and  allowed  to  flow  under.  The  formation  of  crystals  begins 
where  the  solution  and  the  nitric  acid  meet.  Alkali  nitrates  may  crystallize 
very  similarly  to  urea  nitrate  when  they  are  contaminated  with  other  bodies; 
therefore,  in  testing  for  urea,  the  crystals  must  be  identified  as  urea  nitrate  by 
heating  and  by  other  means. 

Urea  Oxalate,  2.CO(NH2)2.H2C204.  This  compound  is  more  spar- 
ingly soluble  in  water  than  the  nitric-acid  compound.  It  is  obtained  in 
rhombic  or  six-sided  prisms  or  plates  on  adding  a  saturated  oxalic-acid 
solution  to  a  concentrated  solution  of  urea. 

Urea  also  forms  combinations  with  mercuric  nitrate  in  variable  propor- 
tions. If  a  very  faintly  acid  mercuric-nitrate  solution  is  added  to  a  2 
per  cent  solution  of  urea  and  the  mixture  carefully  neutralized,  a  com- 
bination is  obtained  of  a  constant  composition  which  contains  for  every 
10  parts  of  urea  72  parts  of  mercuric  oxide.  This  compound  serves  as  the 
basis  of  Liebig's  titration  method.  Urea  combines  also  with  salts,  forming 
mostly  crystallizable  combinations,  as,  for  instance,  with  sodium  chloride, 
with  the  chlorides  of  the  heavy  metals,  etc.  An  alkaline  but  not  a  neutral 
solution  of  urea  is  precipitated  with  mercuric  chloride. 

If  urea  is  dissolved  in  dilute  hydrochloric  acid  and  then  an  excess  of  formal- 
dehyde is  added,  a  thick,  white,  granular  precipitate  is  obtained  which  is  difficultly 
soluble  and  whose  composition  is  somewhat  disputed.1  With  phenylhydrazine, 
urea  in  strong  acetic  acid  gives  a  colorless  crystalline  combination  of  phenyl- 
semicarbazid,  C6H5NH.NH.CONH2,  which  is  soluble  with  difficulty  in  cold  water 
and  melts  at  172°  C.  (Jaffe  2). 

The  method  of  preparing  urea  from  urine  is  in  the  main  as  follows :  Con- 
centrate the  urine,  which  has  been  faintly  acidified  with  sulphuric  acid,  at  a 
low  temperature,  add  an  excess  of  nitric  acid,  at  the  same  time  keeping  the 
mixture  cool,  press  the  precipitate  well,  decompose  it  in  water  with  freshly 


»See  Tollens  and  his  pupils,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29,  2751;    Gold- 
schmidt,  ibid.,  29,  and  Chem.  Centralbl.,  1897,  1,  33;  Thorns,  ibid.,  2,  144  and  737. 
'Zeitschr.  f.  physiol.  Chem.,  22. 


QUANTITATIVE  ESTIMATION  OF    UREA.  475 

precipitated  barium  carbonate,  dry  on  the  water-bath,  extract  the  residue 
with  Btrong  alcohol,  decolorize  when  necessary  with  animal  charcoal,  and 
filter  while  warm.  The  urea  which  crystallizes  on  cooling  is  purified  by 
recrystallization  from  warm  alcohol.  A  further  quantity  of  urea  may  be 
obtained  from  the  mother-liquor  by  concentration.  The  urea  is  purified 
from  contaminating  mineral  bodies  by  redissolving  in  alcohol-ether.  If  it 
is  only  necessary  to  detect  the  presence  of  urea  in  urine,  it  Is  sufficient  to 
concentrate  a  little  of  the  urine  on  a  watch-glass  and,  after  cooling,  treat  it 
with  an  excess  of  nitric  acid.     In  this  way  we  obtain  crystals  of  urea  nitrate. 

Quantitative  Estimation  of  the  Total  Nitrogen  and  Urea  in  Urine.  Among 
the  various  methods  proposed  for  the  estimation  of  the  total  nitrogen,  that 
suggested  by  Kjeldahl  is  to  be  recommended.  But  as  Liebig's  method 
for  the  estimation  of  urea  is  really  a  method  for  determining  the  total 
nitrogen,  and  as  the  physician  has  not  always  at  hand  the  apparatus  and 
utensils  necessary  for  a  Kjeldahl  determination,  he  often  makes  use  of 
this  method;  hence  both  will  be  given  in  detail. 

Kjeldahl 's  method  consists  in  transforming  all  the  nitrogen  of  the 
organic  substances  into  ammonia  by  heating  with  a  sufficiently  concentrated 
sulphuric  acid.  The  ammonia  is  distilled  off  after  supersaturating  with 
alkali  and  the  ammonia  collected  in  standard  sulphuric  acid.  The  follow- 
ing reagents  are  necessary. 

1.  Sulphuric  Acid.  Either  a  mixture  of  equal  volumes  of  pure  concen- 
trated and  fuming  sulphuric  acid  or  else  a  solution  of  200  grams  phosphoric 
anhydride  in  1  liter  of  pure  concentrated  sulphuric  acid.  2.  Caustic  soda  free 
from  nitrates,  30-40  per  cent  solution.  The  quantity  of  this  caustic-soda 
solution  necessary  to  neutralize  10  c.  c.  of  the  acid  mixture  must  be  deter- 
mined. 3.  Metallic  mercury  or  pure  yellow  mercuric  oxide.  (The  addition  of 
this  facilitates  the  destruction  of  the  organic  substances.)  4.  A  potassium- 
sulphide  solution  of  4  per  cent,  whose  object  is  to  decompose  any  mercuric 
amid  combination  which  might  not  have  evolved  its  ammonia  completely 
during  the  distillation  with  caustic  soda.  5.  N/5  sulphuric  acid  and  N/5 
caustic  potash  solution. 

In  performing  the  determination  5  c.  c.  of  the  carefully  measured  and  fil- 
tered urine  is  placed  in  a  long-neck  Kjeldahl  flask,  a  drop  of  mercury  or 
about  0.3  gram  of  mercuric  oxide  added,  and  then  treated  with  10-15  c.  c.  of 
the  strong  sulphuric  acid.  The  contents  are  heated  very  carefully,  placing  the 
flask  at  an  angle,  until  they  just  begin  to  boil  gently;  this  is  continued  for 
about  half  an  hour  after  the  mixture  becomes  colorless.  On  cooling  the  con- 
tents are  transferred  to  a  voluminous  distilling-flask,  carefully  washing  the 
Kjeldahl  flask  with  water,  and  the  greater  part  of  the  acid  is  neutralized 
by  caustic  soda.  A  few  zinc  shavings  arc  added  to  prevent  too  rapid 
ebullition  on  distillation,  and  then  an  excess  of  caustic-soda  solution  which 
has  previously  been  treated  with  30—40  c.  c.  of  the  potassium-sulphide  solu- 
tion. The  flask  is  quickly  connected  with  the  condenser-tube  and  all  the 
ammonia  distilled  off.  In  order  to  prevent  loss  of  ammonia  it  is  best  to 
lower  the  end  of  the  exit-tube  below  the  surface  of  the  acid,  and  the  regur- 
gitation of  the  acid  is  prevented  by  having  a  bulb  blown  on  the  exit-tube. 
Not  less  than  25-30  c.  c.  of  the  standard  acid  is  used  for  every  5  c.  c.  of 
urine,  and  on  completion  of  the  distillation  the  acid  is  ret  it  rated  with  X  5 
caustic  soda,  using  rosolic  acid,  tincture  of  cochineal,  or  lacmoid  as 
indicator.  Each  cubic  centimetre  of  the  acid  corresponds  to  2.8  milligrams 
nitrogen.     As  a  control  and  in  order  to  see  the  purity  of  the  reagents,  or  to 


476  URINE. 

i 

eliminate  any  error  caused  by  an  accidental  quantity  of  ammonia  in  the 
air,  we  always  make  a  blank  determination  with  the  reagents. 

Liebig's  method  is  based  upon  the  fact  that  a  dilute  solution  of  mer- 
curic nitrate  under  proper  conditions  precipitates  all  the  urea  from  its 
solution,  forming  a  compound  of  constant  composition.  As  indicator,  a 
soda  solution  or  a  thin  paste  of  sodium  bicarbonate  is  used.  An  excess  of 
mercuric  nitrate  produces  herewith  a  yellow  or  yellowish-brown  combina- 
tion, while  the  combination  of  urea  and  mercury  is  white.  Pfluger  *  has 
given  full  particulars  of  this  method;  therefore  we  will  describe  Pfluger' s 
modification  of  Liebig's  method. 

As  phosphoric  acid  is  also  precipitated  by  the  mercuric-nitrate  solution,  this 
must  be  removed  from  the  urine  by  the  addition  of  a  baryta  solution  before  titra- 
tion. Pfluger  also  suggested  that  the  acidity  produced  by  the  mercury  solution 
be  neutralized  during  titration  by  the  addition  of  a  soda  solution.  The  liquids 
necessary  for  the  titration  are  the  following: 

1.  Mercuric-nitrate  Solution.  This  solution  is  calculated  for  a  2  per  cent  urea 
solution,  and  20  c.  c.  of  the  first  should  correspond  to  10  c.  c.  of  the  latter.  Each 
cubic  centimeter  of  the  mercury  solution  corresponds  to  0.01  gram  urea.  As  a 
small  excess  of  HgO  is  necessary  in  the  urine  to  cause  the  final  reaction  (with 
alkali  carbonate  or  bicarbonate)  to  appear,  each  cubic  centimeter  of  the  mercury 
solution  must  contain  0.0772  instead  of  0.0720  gram  HgO.  The  mercury  solution 
contains  therefore  77.2  grams  HgO  in  1  liter. 

The  solution  may  be  prepared  from  pure  mercury  or  mercuric  oxide  by  dis- 
solving in  nitric  acid.  The  solution,  freed  as  completely  as  possible  from  an 
excess  of  acid,  is  diluted  by  the  careful  addition  of  water,  stirring  meanwhile, 
until  it  has  a  specific  gravity  of  1.10,  or  a  little  higher,  at  20°  C.  The  solution 
is  standardized  with  a  2  per  cent  solution  of  pure  urea  which  has  been  dried  over 
sulphuric  acid  and  the  operation  conducted  as  will  be  described  later.  If  the 
solution  is  too  concentrated,  it  is  corrected  by  the  careful  addition  of  the  necessary 
amount  of  water,  avoiding  the  precipitation  of  the  basic  salt  and  titrating  again. 
The  solution  is  correct  if  19.8  c.  c.  of  it,  added  at  once  to  10  c.  c.  of  the  urea 
solution  and  the  quantity  (11-12  c.  c.  or  more)  of  normal  soda  solution  necessary 
to  nearly  completely  neutralize  the  liquid,  gives  the  final  reaction  when  exactly 
20  c.  c.  of  the  mercury  solution  has  been  employed. 

2.  Baryta  Solution.  This  consists  of  1  vol.  of  barium  nitrate  and  2  vols,  of 
barium-hydrate  solution,  both  saturated  at  the  ordinary  temperature. 

3.  Normal  Soda  Solution.  This  solution  contains  53  grams  of  pure  anhydrous 
sodium  carbonate  in  1  litre  of  water.  According  to  Pfluger  a  solution  having 
a  specific  gravity  of  1.053  is  sufficient.  The  amount  of  this  soda  solution  neces- 
sary to  completely  neutralize  the  acid  set  free  during  the  titration  is  determined 
by  titrating  with  a  pure  2  per  cent  urea  solution.  To  facilitate  operations  a  table 
can  be  made  showing  the  quantity  of  coda  solution  necessary  when  from  10  to 
35  c.  c.  of  the  mercury  solution  is  used. 

Before  the  titration  the  following  must  be  considered.  The  chlorides  of 
the  urine  interfere  with  the  titration  in  that  a  part  of  the  mercuric  nitrate 
is  transformed  into  mercuric  chloride,  which  does  not  precipitate  the  urea. 
The  chlorides  of  the  urine  are  therefore  removed  by  a  silver-nitrate  solu- 
tion, which  also  removes  any  bromine  or  iodine  combinations  which  may 
exist  in  the  urine.  If  the  urine  contains  proteid  in  noticeable  amounts,  it 
must  be  removed  by  coagulation  and  the  addition  of  acetic  acid,  but  care 
must  be  taken  that  the  concentration  and  the  volume  of  the  urine  are  not 
changed  during  these  operations.     If  the  urine  contains  ammonium  car- 

1  Pfluger,  and  Pfluger  and  Bohland,  in  Pfluger 's  Arch.,  21,  36,  37,  and  40. 


LIEBICP8  TITRATION  METHOD.  177 

bonatc  in  noticeable  quantities,  caused  by  alkaline  fermentation,  this  titra- 
tion method  cannot  be  applied.    The  same  is  true  of  urine  containing  leucin, 

tyrosin,  <»r  medicinal  preparations  precipitated  by  mercuric  nitrate. 

In  cases  where  the  urine  is  free  from  proteld  or  sugar  and  not  specially 
poor  in  chlorides,  the  quantity  of  urea,  and  also  the  approximate  quantity 
of  mercuric  nitrate  necessary  for  the  titration,  may  be  learned  from  the 
specific  gravity.  A  specific  gravity  of  1.010  corresponds  to  aboul  H)  p.  m., 
a  specific  gravity  of  1.015  generally  somewhat  less  than  15  p.  m.,  and  a 
specific  gravity  of  1.015-1.020  about  15-20  p.  m.  urea.  With  a  specific 
gravity  higher  than  1.020  the  urine  generally  contains  more  than  20  p.  in. 
of  urea,  and  above  this  point  the  amount  of  urea  increases  much  more 
rapidly  than  the  specific  gravity,  so  that  with  a  specific  gravity  of  1.030  it 
contains  over  40  p.  m.  urea.  Fever-urines  with  a  specific  gravity  above 
1.020  sometimes  contain  30-40  p.  m.  urea,  or  even  more. 

Preparation  for  the  Titration.  If  a  large  amount  of  urea  is  sus- 
pected from  a  high  specific  gravity,  the  urine  must  first  be  diluted  with  a 
carefully  measured  quantity  of  wrater,  so  that  the  amount  of  urea  is  re- 
duced below  30  p.  m.  In  a  special  portion  of  the  same  urine  the  amount  of 
chlorides  is  determined  by  one  of  the  methods  which  will  be  given  later,  and 
the  number  of  cubic  centimetres  of  silver-nitrate  solution  necessary  is 
noted.  Then  a  larger  quantity  of  urine,  say  100  c.  c,  is  mixed  with  one- 
half  or,  if  this  is  not  sufficient  to  precipitate  all  the  sulphuric  and  phos- 
phoric acids,  with  an  equal  volume  of  the  baryta  solution;  it  is  then  allowed 
to  stand  a  little  while,  and  the  precipitate  is  filtered  through  a  dried  filter. 
From  the  filtrate  containing  the  urine  diluted  with  water  a  proper  quan- 
tity, corresponding  to  about  60  c.  c.  of  the  original  urine,  is  measured,  and 
exactly  neutralized  with  nitric  acid  added  from  a  burette,  so  that  the  exact 
quantity  employed  is  known.  The  neutralized  mixture  of  urine  and  baryta 
is  treated  with  the  proper  quantity  of  silver-nitrate  solution  necessary  to 
completely  precipitate  the  chlorides,  which  were  ascertained  by  a  previous 
determination.  The  mixture,  containing  a  known  volume  of  urine,  is  now 
filtered  through  a  dried  filter  into  a  flask,  and  from  the  filtrate  an  amount 
is  measured  off  corresponding  to  10  c.  c.  of  the  original  urine. 

Execution  of  the  Titration.  Nearly  the  whole  quantity  of  the  mer- 
curic-nitrate solution,  which  is  judged  from  the  specific  gravity  of  the  urine 
to  be  the  minimum  amount  required,  is  added  at  once,  and  immediately 
afterwards  the  quantity  of  soda  solution  necessary,  as  indicated  by  the 
table.  If  the  mixture  becomes  yellowish  in  color,  then  too  much  mercury 
solution  has  been  added  and  another  determination  must  be  made.  If  the 
test  remains  white,  and  if  a  drop  taken  out  and  placed  on  a  glass  plate 
with  a  dark  background  and  stirred  with  a  drop  of  a  thin  paste  of  sodium 
bicarbonate  does  not  give  a  yellow  color,  the  addition  of  mercury  solution  Ls 
continued  by  adding  4  and  then  T17  c.  c,  and  testing  after  each  addition  in 
the  following  way:  A  drop  of  the  mixture  is  placed  on  a  glass  plate  with  a 
dark  background  beside  a  small  drop  of  the  bicarbonate  paste.  If  the  color 
after  stirring  the  two  drops  together  is  still  white  after  a  few  seconds,  then 
more  mercury  solution  must  be  added;  if,  on  the  contrary,  it  Ls  yellowish, 
then — if  not  too  much  mercury  solution  has  been  added  by  inattention — 
the  result  to  Ty  c.  c.  has  been  found.  By  this  approximate  determination, 
which  Is  sufficient  in  many  cases,  we  have  fixed  the  minimum  amount  of 
mercury  solution  necessary  to  add  to  the  quantity  of  urine  in  question, 
and  we  now  proceed  to  the  final  determination.  * 


47S  URINE. 

A  second  quantity  of  the  filtrate,  corresponding  to  10  c.  c.  of  the 
original  urine,  is  filtered,  and  the  same  quantity  of  mercury  solution 
added  at  one  time  as  was  found  necessary  to  produce  the  final  reaction, 
and  immediately  after  the  corresponding  amount  of  soda  solution, 
which  must  not  indicate  the  end  of  the  reaction.  Then  continue  adding 
the  mercury  solution  TV  c.  c.  at  a  time  without  neutralizing  with  soda, 
until  a  drop  taken  out  and  mixed  with  the  soda  solution  gives  a 
yellow  coloration.  If  this  final  reaction  is  obtained  after  the  addition 
of  0.1-0.2  c.  c,  then  the  titration  may  be  considered  as  finished.  If, 
on  the  contrary,  a  larger  quantity  is  necessary,  the  addition  of  the  mer- 
cury solution  must  be  continued  until  a  final  reaction  is  obtained  with 
simple  carbonate,  and  the  titration  repeated  again,  adding  the  quan- 
tity of  mercury  solution  used  in  the  previous  test  at  one  time,  and 
also  adding  the  corresponding  amount  of  soda  solution.  If  then  the  end 
reaction  is  obtained  by  the  addition  of  TV  c.  c,  the  titration  may  be 
considered  as  finished. 

If  in  each  titration  a  quantity  of  filtrate  containing  urine  and  baryta 
corresponding  to  10  c.  c.  of  the  original  urine  is  used,  then  the  calculations 
are  very  simple,  since  1  c.  c.  of  mercuric-nitrate  solution  corresponds  to 
0.01  gram  of  urea.  As  the  mercury  solution  is  made  for  a  2  per  cent  urea 
solution,  and  as  the  filtrate  of  urine  and  baryta  generally  contains  less 
urea  (if  the  quantity  of  urea  is  above  2  per  cent,  it  is  easy  to  avoid  any  mis- 
take by  diluting  the  urine  at  the  beginning  of  the  operation),  a  mistake 
occurs  here  which  can  be  corrected  in  the  following  way,  according  to 
Pfluger:  To  the  measured  volume  of  the  filtrate  from  the  urine  (the 
filtrate  with  baryta  after  neutralization  with  nitric  acid,  precipitation  with 
silver  nitrate  and  filtration)  the  quantity  of  normal  soda  solution  employed 
is  added,  and  from  this  sum  the  volume  of  mercury  solution  used  is  sub- 
tracted. The  remainder  is  then  multiplied  by  0.08,  and  the  product  sub- 
tracted from  the  number  of  cubic  centimeters  of  mercury  solution  used. 
For  example,  if  the  filtrate  (urine  and  baryta  +  nitric  acid  +  silver  nitrate) 
measured  25.8  c.  c,  and  the  number  of  cubic  centimeters  of  soda  solution 
used  in  the  titration  was  13.8  c.  c,  and  of  the  mercury  solution  20.5  c.  c,  we 
have  then  20.5 -[(39.6 -20.5)  X0.08]  =  20.5 -1.53  =  18.97,  and  the  corrected 
quantity  of  mercury  solution  is  therefore  18.97  c.  c.  If  the  cubic  centi- 
meters of  the  filtrate  (in  this  case  25.8  c.  c.)  correspond  to  10  c.  c.  of 
the  original  urine,  then  the  amount  of  urea  is  18.97X0.01  =  0.1897=18.97 
p.  m.  urea. 

Besides  the  urea  other  nitrogenous  constituents  of  the  urine  are  precipi- 
tated by  the  mercury  solution.  In  the  titration  we  really  do  not  obtain 
the  quantity  of  urea,  but,  as  Pfluger  has  shown,  the  total  quantity  of 
nitrogen  in  the  urine  expressed  as  urea.  As  urea  contains  46.67  per  cent  N, 
the  total  quantity  of  nitrogen  in  the  urine  may  be  calculated  from  the 
quantity  of  urea  found.  The  results  obtained  by  this  calculation  cor- 
respond  well,  according  to  Pfluger,  with  the  results  found  for  the  total 
nitrogen  as  determined  by  Kjeldahl's  method. 

Among  the  methods  suggested  for  the  special  estimation  of  urea,  that  of 
MoRNER-SJOQVIST,  in  combination  with  Folin's  method,  is  perhaps  the 
most  trustworthy  and  readily  performed.  For  this  reason  this  method  only 
will  be  given  in  detail,  -while  we  must  refer  to  special  works  for  the  other 


METHODS  FOR  ESTIMATING   UREA.  479 

methods,  such  as  BuNSEN'fl  method  with  its  many  modificatioas  as  sug- 
1  by  Pflugbr,  Bohland  and  Bleibtreu.1 

Principle  of  Morner-Sjoqvist'a  Method.1  According  to  this  method  the 
nitrogenous  constituents  of  the  urine,  with  the  exception  of  urea,  ammonia> 
hippuric  acid,  creatinine,  and  traces  of  allantoin  are  precipitated  by  a  mix- 
ture of  alcohol  and  ether  after  the  addition  of  a  solution  of  barium  chloride 
and  barium  hydrate  or  in  the  presence  of  sugar  with  solid  barium  hydrate. 
The  urea  is  determined  in  the  concentrated  filtrate,  after  driving  off  the 
ammonia,  by  K.ikldahl's  nitrogen  estimation.  Because  of  the  alight  error 
due  to  the  presence  of  hippuric  acid  and  creatinine,  several  modifications 
have  been  suggested  by  Salaskin  and  Zaleski  and  by  Brauxstkiv3 
These  errors  are  best  prevented,  according  to  Morxer,  by  the  use  of 
Folix's  method. 

Principle  of  Folin's  Method.*  On  heating  urea  with  hydrochloric  acid 
and  crystalline  magnesium  chloride,  which  melts  in  its  water  of  crystalliza- 
tion at  112-115°  C,  and  then  boils  at  about  150-155°  C,  the  urea  is  com- 
pletely decomposed,  while  no  mentionable  decomposition  of  the  hippuric 
acid  and  creatinine  takes  place.  The  ammonia  produced  from  the  urea  is 
distilled  off  and  determined  by  titration.  The  amount  of  ammonia  pre- 
viously existing  in  the  urine  must  be  specially  determined. 

Determination  of  Urea  by  the  Mdrner-Sjoqvist  and  Folin  Method.5  Fivec.  c. 
of  the  urine  are  treated  with  1.5  grams  of  powdered  barium  hydroxide,  and 
when  as  much  of  this  is  dissolved  as  possible  by  gently  mixing,  it  is, pre- 
cipitated by  100  c.  c.  of  the  alcohol  and  ether  mixture  (£  vol.  ether).  On 
the  following  day  it  is  filtered  and  the  precipitate  washed  with  the  alcohol 
and  ether  mixture.  The  alcohol  and  ether  are  distilled  off  from  the  filtrate 
at  about  55°  C.  (not  above  60°  C).  The  remaining  liquid  is  treated  with 
2  c.  c.  of  hydrochloric  acid  of  sp.  gr.  1.124  (for  5  c.  c.  urine),  and  carefully 
transferred  to  a  flask  of  200  c.  c.  capacity,  and  evaporated  to  dryness  on 
the  water-bath.  Then  add  20  grams  of  crystalline  magnesium  chloride  to 
the  contents  of  the  flask  and  2  c.  c.  of  concentrated  hydrochloric  acid,  and  boil 
on  a  wire  gauze  over  a  small  flame  for  two  hours,  making  use  of  a  proper 
return  cooler.  After  cooling  it  is  diluted  to  about  f  to  1  liter  of  water,  the 
ammonia  completely  distilled  off  after  making  it  alkaline  with  caustic  soda, 
and  the  ammonia  collected  in  standard  acid.  After  boiling  in  order  to  drive 
off  the  CO,  and  cooling,  the  acid  is  retitrated.  Corrections  must  be  made  for 
the  ammonia  of  the  urine  and  for  that  contained  in  the  magnesium  chloride. 

If  a  special  determination  of  the  preformed  ammonia  has  been  made,  then  a 
direct  treatment  of  the  urine  according  to  Folin  (nevertheless  after  the  evapo- 
ration of  the  urine  with  hydrochloric  acid)  gives  good  results.     In  the  presence  of 

1  Pfliiger's  Arch.,  3S,  43,  and  44. 

:  Skand.  Arch.  f.  Physiol.,  2,  and  Morner,  ibul.,  14,  where  the  recent  literature 
may  also  be  found. 

3  Braunstein,  Zeitschr.  f.  physiol.  Chem.,  31;   Salaskin  and  Zaleski,  ibid.,  28. 
4Zeitschr.  f.  physiol.  Chem.,  32,  36,  and  37. 
5  See  Morner,  Skand.  Arch.  f.  Physiol.,  14. 


480  URINE. 

sugar  the  treatment  of  the  urine  with  barium  hydroxide  is  absolutely  necessary 
according  to  Morner,  otherwise  the  humin  substances  produced  from  the  sugar 
take  up  and  retain  nitrogen. 

Knop-Hufner  's  method  l  is  based  on  the  fact  that  urea,  by  the  action  of 
sodium  hypobromite,  splits  into  water,  carbon  dioxide  (which  dissolves  in  the 
alkali),  and  nitrogen,  whose  volume  is  measured  (see  page  473).  This  method 
is  less  accurate  than  the  preceding  ones,  and  therefore  in  scientific  work  it  is 
discarded.  It  is  of  value  to  the  physician  and  for  practical  purposes,  because 
of  the  ease  and  rapidity  with  which  it  may  be  performed,  even  though  it  may 
not  give  very  accurate  results.  For  practical  purposes  a  series  of  different  appa- 
ratus have  been  constructed  to  facilitate  the  use  of  this  method. 

For  the  quantitative  estimation  of  urea  in  blood  or  other  animal  fluids, 
as  well  as  in  the  tissues,  Schondorff  has  proposed  a  method  where  the 
proteids  and  extractives  are  first  precipitated  by  a  mixture  of  phospho- 
tungstic  acid  and  hydrochloric  acid,  and  then  the  filtrate  made  alkaline 
with  lime.  The  quantity  of  ammonia  formed  on  heating  a  part  of  this 
filtrate  to  150°  C.  with  phosphoric  acid  and  the  amount  of  carbon  dioxide 
produced  by  heating  the  other  part  to  150°  C.  are  determined.  In  regard 
to  the  principles  of  this  method,  as  well  as  to  the  details,  we  refer  to  the 
original  article  (Pfluger's  Arch.,  62).  See  also  Hoppe-Seyler-Thier- 
felder's  Handbuch.,  7.  Aufl. 

Urein  is  the  name  given  by  Ovid  Moor  to  a  product  which  he  obtained  by 
extracting  the  urine,  which  had  been  evaporated  to  a  syrup,  with  absolute  alcohol 
and  precipitating  the  urea  with  alcohol  containing  oxalic  acid,  or  by  cooling  and 
treatment  with  alcohol.  Urein  is  a  golden-yellow  oil  which  is  poisonous;  it  re- 
duces permanganate  in  the  cold  and  it  forms  the  chief  portion  of  the  nitrogenous 
extractives  of  the  urine.  There  is  no  doubt  but  that  urein  is  a  mixture  of  sub- 
stances. According  to  Moor,2  the  amount  of  urea  in  the  urine  is  only  about 
one  half  that  ordinarily  given,  and  he  has  suggested  a  new  method  for  the  deter- 
mination of  the  true  quantity  of  urea.  The  possibility  that  in  the  urine  we  have 
other  bodies  besides  urea  which  have  been  determined  with  the  urea  must  not 
be  denied  a  priori.  From  the  investigations  published  thus  far  it  must  be  said 
that  Moor's  assertions  are  not  sufficiently  grounded.3 

■NT  XT 

Carbamic  Acid,  CH3X02  =  CO  <  qtt2.     This  acid  is  not  known  in  the  free  state, 

but  only  as  salts.  Ammonium  carbamate  is  produced  by  the  action  of  dry  ammo- 
nia on  dry  carbon  dioxide.  Carbamic  acid  is  also  produced  by  the  action  of 
potassium  permanganate  on  proteid  and  several  other  nitrogenous  organic  bodies. 
The  occurrence  of  carbamic  acid  in  human  and  animal  urines  has  already 
been  considered  in  connection  with  the  formation  of  urea.  The  calcium  salt,  which 
is  soluble  in  water  and  ammonia  but  insoluble  in  alcohol,  is  the  most  important 
in  the  detection  of  this  acid.  The  solution  of  the  calcium  salt  in  water  becomes 
cloudy  on  standing,  but  much  quicker  on  boiling,  and  calcium  carbonate  sepa- 
rates. Nolf  '  has  made  investigations  on  the  formation  and  detection  of  car- 
bamic acid,  which  question  the  special  physiological  origin  of  carbamic  acid. 

1  Knop,  Zeitschr.  f.  analyt.  Chem.,  9;  Hiifner,  Journ.  f.  prakt.  Chem.  (N.  F.),  3. 
In  regard  to  the  extensive  literature,  see  Huppert-Neubauer,  10.  Aufl.,  304,  and  follow- 
ing. 

2  O.  Moor,  Bull.  Acad,  de  St.  P6tersbourg,  14  (also  Maly's  Jahresber.,  31,  415); 
and  Zeitschr.  f.  Biologie,  44,  and  Zeitschr.  f.  physiol.  Chem.,  40. 

3  See  Kubiabko,  Maly's  Jahresber.,  31,  415;  Erben.,  Zeitschr.  f.  physiol.  Chem., 
38;    Folin,  ibid.,  37. 

*  Zeitschr.  f.  physiol.  Chem.,  23. 


CREATININE.  481 

Carbamic  acid  ethylestcr  (urethan),  as  shown  by  Jaffe,1  may  pass,  by  the 
mutual  action  of  alcohol  and  urea,  into  the  alcoholic  extract  of  the  urine  when 
working  with  large  quantities  of  urine. 

,NH CO 

Creatinine,  CJLN.O,  or  XH:C<f  I      ,  is  generally  considered  as 

\\^CH3).CH2 

the  anhydride  of  creatine  (see  page  383)  found  in  the  muscles.  It  occurs 
in  human  urine  and  in  that  of  certain  mammalia.  It  has  also  been  found 
in  ox-blood,  milk,  though  in  very  small  amounts,  and  in  the  flesh  of  certain 
fishes. 

Johnson's  statement  that  the  creatinine  of  the  urine  is  different  from  that  pro- 
duced by  the  action  of  acids  on  creatine  is  incorrect  according  to  Toppelius  and 

POMMEREHNE,    WoERNER    and    ThELEN.2 

The  quantity  of  c  eatinine  in  human  urine  is,  in  a  grown  man  voiding  a 
normal  quantity  of  urine  in  the  course  of  a  day,  0.6-1.3  grams  (Neubauer), 
or  on  an  average  1  gram.  Johnson  3  found  1.7-2.1  grams  per  day.  The 
quantity  is  dependent  on  the  food  and  decreases  in  starvation.  Sucklings 
do  not  generally  eliminate  any  creatinine,  and  it  only  appears  in  the  urine 
when  the  milk  is  replaced  by  other  food.  The  quantity  of  creatinine  in 
urine  varies  as  a  rule  with  the  quantity  of  urea,  although  it  is  increased 
more  by  meat  (because  the  meat  contains  creatine)  than  by  proteid.  Grocco, 
Moitessier,  and  Gregor  claim  that  the  elimination  of  creatinine  is  in- 
creased by  muscular  activity,  but  according  to  Oddi  and  Tarulli  4  this  is 
only  true  for  excessive  activity.  The  behavior  of  creatinine  in  disease  is 
little  known.  By  increased  metabolism  the  amount  is  increased,  while  by 
decreased  exchange  of  material,  as  in  anaemia  and  cachexia,  it  is 
diminished. 

Creatinine  crystallizes  in  colorless,  shining  monoclinic  prisms  which 
differ  from  creatine  crystals  in  not  becoming  white  with  loss  of  water  when 
heated  to  100°  C.  It  dissolves  in  11  parts  cold  water,  but  more  easily  in 
warm  water.  It  is  difficultly  soluble  in  cold  alcohol,  but  the  statements 
in  regard  to  its  solubilities  differ  widely.8  It  is  more  soluble  in  warm  alcohol 
and  nearly  insoluble  in  ether.  In  alkaline  solution  creatinine  is  converted 
into  creatine  very  easily  on  warming. 

Creatinine  gives  an  easily  soluble  crystalline  combination  with  hydro- 
chloric acid.     A  solution  of  creatinine  acidified  with  mineral  acids  gives 

1  Zeitschr.  f.  physiol.  Chem.,  14. 

*  S.  Johnson,  Proceed.  Roy.  Soc.,  42,  43;  Chem.  News,  55;  Toppelius  and  Pom- 
merehne,  Arch.  f.  Pharm.,  234;   Woerner,  Du  Bois-Reymond 's  Arch.,  1898. 

3  Huppert-Xeubauer,  Harnanalyse,  10.  Aufl.,  387. 

4  Grocco,  see  Maly's  Jahresber.,  16;  Moitessier,  ibid.,  21;  Oddi  and  Tarulli,  ibid., 
24;   Gregor,  Zeitschr.  f.  physiol.  Chem.,  31. 

s  See  Huppert-Xeubauer,  10.  Aufl.,  and  Hoppe-Seyler-Thierf elder 's  Handbuch, 
7.  Aufl. 


4S2  URINE. 

crystalline  precipitates  with  phosphotungstic  or  phosphomolybdic  acids 
even  in  very  dilute  solutions  (1:10,000)  (Kerner,  Hofmeister  *) .  It  is 
precipitated,  like  urea,  by  mercuric-nitrate  solution  and  also  by  mercuric 
chloride.  On  treating  a  dilute  creatinine  solution  with  sodium  acetate  and 
then  with  mercuric  chloride  a  precipitate  of  glassy  globules  having  the 
composition  4(C4H7N3O.HCl.HgO)3HgCl2  separates  on  standing  some  time 
(Johnson).  Among  the  compounds  of  creatinine,  that  with  zinc  chloride, 
creatinine  zinc-chloride,  (C4H7N30)2ZnCl2,  is  of  special  interest.  This  com- 
bination is  obtained  when  a  sufficiently  concentrated  solution  of  creati- 
nine in  alcohol  is  treated  with  a  concentrated,  faintly  acid  solution  of 
zinc  chloride.  Free  mineral  acids  dissolve  the  combination,  hence  they 
must  not  be  present;  this,  however,  may  be  prevented  by  an  addition  of 
sodium  acetate.  In  the  impure  state,  as  ordinarily  obtained  from  urine, 
creatinine  zinc-chloride  forms  a  sandy,  yellowish  powder  which  under  the 
microscope  appears  as  fine  needles  forming  concentric  groups,  mostly  com- 
plete rosettes  or  yellow  balls  or  tufts,  or  grouped  as  brushes.  On  slowly 
crystallizing  or  when  very  pure,  more  sharply  defined  prismatic  crystals 
are  obtained.     This  combination  is  sparingly  soluble  in  water. 

Creatinine  acts  as  a  reducing  agent.  Mercuric  oxide  is  reduced  to 
metallic  mercury,  and  oxalic  acid  and  methylguanidine  (methyluramine) 
are  formed.  Creatinine  also  reduces  cupric  hydrate  in  alkaline  solution, 
forming  a  colorless  soluble  combination,  and  only  after  continuous  boiling 
with  an  excess  of  copper  salt  is  free  suboxide  of  copper  formed.  Creatinine 
interferes  with  Trommer's  test  for  sugar,  partly  because  it  has  a  reducing 
action  and  partly  by  retaining  the  copper  suboxide  in  solution.  The  com- 
bination with  copper  suboxide  is  not  soluble  in  a  saturated  soda  solution,  and 
if  a  little  creatinine  is  dissolved  in  a  cold  saturated  soda  solution  and 
then  a  few  drops  of  Fehling  's  reagent  added,  a  white  flocculent  combina- 
tion separates  after  heating  to  50-60°  C.  and  then  cooling  (v.  Maschke's  2 
reaction).  An  alkaline  bismuth  solution  (see  Sugar  Tests)  is  not  reduced 
by  creatinine. 

If  we  add  a  few  drops  of  a  freshly  prepared  very  dilute  sodium  nitro-prus- 
side  solution  (sp.  gr.  1.003)  to  a  dilute  creatinine  solution  (or  to  the  urine)  and 
then  a  few  drops  of  caustic  soda,  a  ruby-red  liquid  is  obtained  which  quickly 
turns  yellow  again  (Weyl's  3  reaction).  If  the  cold  yellow  solution  is 
neutralized  and  treated  with  an  excess  of  acetic  acid  a  crystalline  precipi- 
tate of  a  nitroso  compound  (C4H6N402)  of  creatinine  separates  on  stirring 
(Kramm  4)-  K>  on  tne  contrary,  the  yellow  solution  is  treated  with  an 
excess  of  acetic  acid  and  heated,  the  solution  becomes  first  green  and  then 


1  Kerner,  Pfliiger's  Arch  ,  2;  Hofmeister,  Zeitschr.  f.  physiol.  Chenx,  5. 

2  Zeitschr.  f.  analyt.  Chem.,  17. 

8  Ber.  d.  deutsch.  chem.  Gesellsch.,  11. 
4Centralbl.  f.  d.  med.  Wissensch.,  1897. 


ESTIMATION  OF  CREATININE.  483 

blue  (Salkowskt  ');  finally  a  precipitate  of  Prussian  blue  is  obtained.  If  a 
solution  of  creatinine  in  water  (or  urine)  is  treated  with  a  watery  solution 
of  picric  acid  and  a  few  drops  of  a  dilute  caustic-soda  solution,  a  red  colora- 
tion lasting  several  hours  occurs  immediately  at  the  ordinary  temperature, 
which  turns  yellow  on  the  addition  of  acid  (Jaffa's2  reaction).  Ace- 
tone gives  a  more  reddish-yellow  color.  Dextrose  gives  wit  h  this  reagent 
a  red  coloration  only  after  heating. 

In  preparing  creatinine  from  urine  the  creatinine  zinc-chloride  is  first 
prepared  according  to  Neubauer  's  3  method.  One  liter  or  more  of  urine  is 
treated  with  milk  of  lime  until  alkaline  and  then  CaCl2  solution  is  added 
until  all  the  phosphoric  acid  is  precipitated.  The  filtrate  is  evaporated  toa 
syrup  after  faintly  acidifying  with  acetic  acid  and  this  treated  while  still  warm 
with  97  per  cent  alcohol  (about  200  c.  c.  for  each  liter  of  urine).  After 
about  twelve  hours  it  is  filtered  and  the  filtrate  treated  first  with  a  little 
sodium  acetate  and  then  with  an  acid-free  zinc-chloride  solution  of  a  specific 
gravity  of  1.20  (about  2  c.  c.  for  each  liter  of  urine).  After  thorough  stir- 
ring it  is  allowed  to  stand  forty-eight  hours,  the  precipitate  collected  on  a 
filter  and  washed  with  alcohol.  The  creatinine  zinc-chloride  is  dissolved 
in  hot  water,  boiled  with  lead  oxide,  filtered,  the  filtrate  decolorized  by 
animal  charcoal,  evaporated  to  dryness,  and  the  residue  extracted  with  strong 
alcohol  (which  leaves  the  creatine  undissolved).  The  alcoholic  extract  is 
evaporated  to  the  point  of  crystallization,  and  the  crystals  purified  by 
recrystallization  from  water. 

Creatinine  may  also  be  prepared  from  urine  by  precipitating  with 
a,  mercuric-chloride  solution  according  to  either  Maly's  or  Johnson's4 
process. 

The  quantitative  estimation  of  creatinine  may  be  performed  according  to 
Neubauer's  method  for  the  preparation  of  creatinine  or  more  simply  by 
Salkowski  \s  b  modification  of  this  method.  240  c.  c.  of  the  urine  freed  from 
proteid  (by  boiling  with  acid)  and  from  sugar  (by  fermentation  with  yeast) 
are  made  alkaline  with  milk  of  lime,  and  precipitated  by  CaCl2  and  made  up 
to  300  c.  c.  250  c.  c.  ( =  200  c.  c.  urine)  of  this  are  measured  off,  neutralized 
or  made  only  faintly  acid  with  acetic  acid  and  evaporated  to  about  I'D  c.  <•.. 
then  thoroughly  stirred  with  an  equal  volume  of  absolute  alcohol,  and 
completely  transferred  to  a  100  c.  c.  flask  which  contains  some  alcohol, 
the  residue  in  the  dish  being  washed  with  alcohol.  On  thorough  shaking 
and  cooling,  the  flask  is  filled  up  to  the  100  c.  c.  mark  with  absolute  alcohol 
and  allowed  to  stand  twenty-four  hours.  80  c.  c.  (  =  160  c.  c.  urine)  of 
the  filtrate  are  collected  in  a  beaker  and  treated  with  0.5-1  c.  c.  of  zinc- 
chloride  solution,  and  the  covered  beaker  is  left  standing  in  a  cool  place 
for  two  or  three  days.  The  precipitate  is  collected  on  a  small  dried  and 
weighed  filter,  using  the  filtrate  to  wash  the  crystals  from  the  beaker. 
After  allowing  the  crystals  to  completely  drain  off,  they  are  washed  with 
a  little  alcohol  until  the  filtrate  gives  no  reaction  for  chlorine,  and  dried  at 


1  Zeitschr.  f.  physiol.  Chem.,  4. 

2  Ibid.,  10. 

3  Ann.  d.  Chem.  u.  Pharm.,  119. 

*  Maly,  Annal.  d.  Chem.  u.  Pharm.,  159;  Johnson,  Proceed.  Roy.  Soc,  43. 

*  Zeitschr.  f.  physiol.  Chem.,  10  and  14. 


4S4  URINE. 

100°  C.  100  parts  of  creatinine  zinc-chloride  contain  62.44  parts  of  creatinine. 
As  the  precipitate  is  never  quite  pure,  the  quantity  of  zinc  must  be  care- 
fully determined,  in  exact  experiments,  by  evaporating  with  nitric  acid, 
heating,  washing  the  oxide  of  zinc  with  water  (to  remove  any  NaCl),  drying, 
heating,  and  weighing.  22.4  parts  zinc  oxide  correspond  to  100  parts 
creatinine  zinc-chloride.  Instead  of  weighing,  the  nitrogen  can  be  deter- 
mined by  Kjeldahl's  method  and  the  creatinine  calculated  from  this. 
In  regard  to  other  methods,  see  the  works  of  Kolisch  and  Gregor.1 

Xanthocreatinine,  C5H10N4O.  This  body,  which  was  first  prepared  from  meat 
extract  by  Gautier,  has  been  found  by  Monari  in  dog's  urine  after  the  injection 
of  creatinine  into  the  abdominal  cavity,  and  in  human  urine  after  several  hours 
of  exhausting  marching.  According  to  Colasanti  it  occurs  to  a  relatively  greater 
extent  in  lion 's  urine.  Stadthagen  2  considers  the  xanthocreatinine  isolated 
from  human  urine  after  strenuous  muscular  activity  as  impure  creatinine. 

Xanthocreatinine  forms  thin  sulphur-yellow  plates,  similar  to  cholesterin, 
which  have  a  bitter  taste.  It  dissolves  in  cold  water  and  in  alcohol,  and  gives 
a  crystalline  combination  with  hydrochloric  acid  and  a  double  compound  with 
gold  and  platinum  chloride.  It  gives  a  combination  with  zinc  chloride,  which 
crystallizes  in  fine  needles.     Xanthocreatinine  has  a  poisonous  action. 

HN— CO 

Uric  Acid,  Ur,  C5H4N403,  2,  6,  8-trioxypurin=  OC     C— NHx 

>CO,  has 
HN— C— NH/ 

been  prepared  synthetically  by  Horbaczewski  by  fusing  urea  and  glycocoll 

or  by  heating  trichlorlactic  acid  amide  with  an  excess  of  urea.     Behrend 

and  Roosen  prepared  it  from  isodialuric  acid  and  urea;   it  is  also  readily 

produced  from  isouric  acid  on  boiling  with  hydrochloric  acid  (E.  Fischer 

and  Tullner),  and  finally  E.  Fischer  and  Ach3  have  prepared  uric  acid 

from  pseudouric  acid  by  heating  with  oxalic  acid  to  145°  C. 

On  strongly  heating  uric  acid  it  decomposes  with  the  formation  of 

urea,   hydrocyanic  acid,  cyanuric   acid,  and   ammonia.     On   heating  with 

concentrated  hydrochloric   acid  in  sealed  tubes  to  170°  C.  it  splits  into 

glycocoll,  carbon  dioxide,  and  ammonia.     By  the  action  of  oxidizing  agents 

splitting   and  oxidation  take   place,   and  either   monoureids   or  diureids 

are    produced.      By  oxidation  with  lead    peroxide,  carbon   dioxide,  oxalic 

acid,    urea,  and    allantoin,  which    last    is    glyoxyldiureid,  are    produced 

(see  below).     By  oxidation  with  nitric  acid  in  the  cold  urea  and  a  mono- 

ureid,   the    mesoxalyl    urea,   or     alloxan,   are    obtained,   C5H4N403-|-0  + 

H20==C4H2N204+(NH2)2CO.     On  warming  with  nitric  acid,  alloxan  yields 

carbon   dioxide,  and   oxalyl  urea,  or   parabanic  acid,  C3H2N203.     By  the 


1  Kolisch,  Centralbl.  f.  innere  Med  ,  1895;  Gregor,  Zeitschr.  f.  physiol.  Chem.,  31. 

2 Gautier,  Bull,  de  l'acad.  de  med.  (2),  5,  and  Bull,  de  la  Soc.  Chem.  (2),  48;  Monari, 
Maly's  Jahresber.,  17;  Colasanti,  Arch.  ital.  d.  Biologie,  15,  Fasc.  3;  Stadthagen, 
Zeitschr.  f.  klin.  Med.,  15. 

3  Horbaczewski,  Monatshefte  f.  Chem.,  6  and  8;  Behrend  and  Roosen,  Ber.  d.  d. 
chem.  Gesellsch.,  21;  Fischer  and  Tullner,  ibid.,  35;  Fischer  and  Ach,  ibid  ,  28. 


URIC  ACID.  485 

addition  of  water  the  parabanic  acid  passes  into  oxaluric  acid,  C^H4N,04, 

trails  of  which  are  found  in  the  urine  and  which  easily  split  into  oxalic 
acid  and  urea.  In  alkaline  solution  uric  acid  may,  by  taking  up  water 
and  oxygen,  be  transformed  into  a  new  acid,  uroxanic  acid,  C5H8N\(  )„, 
which  may  then  be  changed  into  oxonic  acid,  QH^O,.1  Uric  acid  may, 
B£  F.  and  L.  Skstixi  as  well  as  GERABD  have  shown,  undergo  bacterial 
fermentation  with  the  formation  of  urea.  According  to  Ulpiaxi  and 
Cingolani,3  uric  acid  is  quantitatively  split  hereby  into  urea  and  carbon 
dioxide,  according  to  the  equation: 

C5H4N403  +  2H20  +  30=  3C02  +  2CO(XH2)2. 

Uric  acid  occurs  most  abundantly  in  the  urine  of  birds  and  of  scaly 
amphibians,  in  which  animals  the  greater  part  of  the  nitrogen  of  the  urine 
appears  in  this  form.  Uric  acid  occurs  frequently  in  the  urine  of  carniv- 
orous mammalia,  but  is  sometimes  absent;  in  urine  of  herbivora  it  is 
habitually  present,  though  only  as  traces;  in  human  urine  it  occurs  in 
greater  but  still  small  and  variable  amounts.  Traces  of  uric  acid  are  also 
found  in  several  organs  and  tissues,  as  in  the  spleen,  lungs,  heart,  pancreas, 
liver  (especially  in  birds),  and  in  the  brain.  It  habitually  occurs  in  the 
blood  of  birds.  Traces  have  been  found  in  human  blood  under  normal 
conditions.  Under  pathological  conditions  it  occurs  to  an  increased  extent 
in  the  blood  as  in  pneumonia  and  nephritis,  but  especially  in  leucaemia  and 
sometimes  also  in  arthritis.  Uric  acid  also  occurs  in  large  quantities  in 
"chalk-stones,"  certain  urinary  calculi,  and  in  guano.  It  has  also  been 
detected  in  the  urine  of  insects  and  certain  snails,  as  also  in  the  wings  (which 
it  colors  white)  of  certain  butterflies  (Hopkins).3 

The  amount  of  uric  acid  eliminated  with  human  urine  is  subject  to 
considerable  individual  variation,  but  amounts  on  an  average  to  0.7  gram 
per  day  on  a  mixed  diet.  The  ratio  of  uric  acid  to  urea  varies  consider- 
ably with  a  mixed  diet,  but  is  on  an  average  1 :  50-1 :  70.  In  new-born 
infants  and  in  the  first  days  of  life  the  elimination  of  uric  acid  is  relatively 
increased,  and  the  relation  between  uric  acid  and  urea  has  been  found  to 
be  1:6.42-17.1. 

We  used  to  ascribe  an  increasing  action  upon  the  elimination  of  uric 
acid  to  protcid  food,  but  the  investigations  of  Hirschfeld,  Rosexfeld 
and  Orgler,  Sivex,  Buriax  and  Schur/  and  others  have  positively 
proven  that  a  diet  rich  in  proteid  does  not  itself  increase  the  elimination 

Sundwik,  Zeitschr.  f.  physiol.  Chem.,  20. 

Chem.  Centralbl.,  1903,  where  the  other  investigators  are  cited. 
J  Philos.  Trans.  Roy.  Soc.,  186,  B.,  661. 

1  See  the  extensive  review  of  the  literature  in  Wiener,  "Die  Harnsaure"  in  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  I,  1902. 


4S6  URINE. 

of  uric  acid  but  only  according  to  the  amount  of  nucleins  or  purin  bodies 
contained  therein.  The  common  statement  that  the  elimination  of  uric 
acid  is  smaller  with  a  vegetable  diet  than  with  an  animal  diet,  when  the 
quantity  may  be  2  grams  or  more  per  twenty-four  hours,  is  explained  by 
this.1 

The  statements  in  regard  to  the  influence  of  other  circumstances,  as 
also  of  different  substances,  on  the  elimination  of  uric  acid  are  rather 
contradictor}^.  This  is  in  part  due  to  the  fact  that  the  older  investi- 
gators used  an  inaccurate  method  (Heintz),  and  also  that  the  extent  of 
uric-acid  elimination  is  dependent  in  the  first  place  upon  the  individuality. 
Thus  the  statements  in  regard  to  the  action  of  drinking-water  2  and  of 
alkalies  3  are  very  contradictory.  Certain  medicines,  such  as  quinine  and 
atropine,  diminish,  while  others,  such  as  pilocarpine  and  also,  as  it  seems, 
salicylic  acid,4  increase  the  elimination  of  uric  acid. 

Little  is  known  with  positiveness  in  regard  to  the  elimination  of  uric 
acid  in  disease.  In  acute  diseases  with  crises  the  elimination  of  uric  acid  is 
increased  after  the  crisis,  while  the  older  statements  that  the  uric  acid  is 
habitually  increased  in  fevers  has  been  contradicted  by  many.  The  state- 
ments in  regard  to  the  elimination  of  uric  acid  in  gout  and  nephritis  are  also 
uncertain  and  contradictory.  In  leucaemia  the  elimination  is  increased 
absolutely  as  well  as  relatively  to  the  urea  and  the  relationship  between 
the  uric  acid  and  urea  (total  nitrogen  recalculated  as  urea)  may  be  even 
1  : 9,  while  under  normal  conditions,  according  to  different  investigators, 
it  is  1  :  40  to  66  to  100.5 

Formation  of  Uric  Acid  in  the  Organism.  Since  Horbaczewski  first 
showed  that  uric  acid  could  be  produced  by  oxidation  from  the  nuclein  rich 
spleen  pulp  or  nucleins  outside  of  the  body  he  also  showed  that  nucleins 
when  introduced  into  the  animal  body  caused  an  increase  in  the  elimination 
of  uric  acid.  These  observations  have  been  confirmed,  and  at  the  same 
time  developed  by  the  work  of  a  great  number  of  investigators,  and 
we  are  sure  that  uric  acid  can  be  produced  from  purin  bodies  either  outside 
or  inside  the  animal  body  and  also  that  food  rich  in  nucleins  (especially 
the  thymus  gland)  increase  the  elimination  of  uric  acid  and  purin  bases 

1  J.  Ranke,  Beobachtungen  und  Versuche  iiber  die  Ausscheidung  der  Harnsaure, 
etc.  (Miinchen,  1858);  Mares,  Centralbl.  f.  d.  med.  Wissensch.,  1888;  Horbaczewski, 
Wien.  Sitzungsber.,  100,  Abt.  3,  1891.  In  regard  to  the  action  of  various  diets  the 
reader  is  referred  to  the  above-cited  authors,  and  especially  to  A.  Hermann,  Arch.  f. 
klin.  Med.,  43,  and  Camerer,  Zeitschr.  f.  Biologie,  33. 

2  See  Schondorff,  Pfliiger's  Arch.,  46,  which  contains  the  pertinent  literature. 

3  See  Clar,  Centralbl.  f.  d.  med.  Wissench.,  1888;  Haig,  Journ.  of  Physiol.,  8;  and 
A.  Hermann,  Arch,  f   klin.  Med.,  43. 

4  See  Bohland,  cited  from  Maly's  Jahresber.,  20;  Schreiber  and  Zaudy,  ibid.,  30. 

5  In  regard  to  the  extensive  literature  on  the  elimination  of  uric  acid  in  disease 
■we  must  refer  to  special  works  on  internal  diseases. 


FORMATION  OF  URIC  ACID.  487 

(alloxuric  bases  *).  Kutscher  and  Skkmaxx  2  have  oxidized  thymus  nu- 
cleic acid  in  weak  soda  solution  with  calcium  permanganate  and  obtained 
no  uric  acid,  but  only  guanidine  and  urea.     Based  on  these  obeervationa  the 

production  of  uric  acid  by  the  oxidation  of  purin  bases  from  the  nucleins 
is  less  probable.  The  original  view  of  Horbaczkwski.  that  the  nucleins  do 
not  directly  cause  an  increased  elimination  of  uric  acid,  but  indirectly  by 
causing  a  leukocytosis  with  a  following  destruction  of  leucocytes,  lias  been 
nearly  generally  discarded.  At  present  it  Ls  considered  that  a  direct 
formation  of  uric  acid  from  the  nucleins  takes  place  by  the  transformation 
of  the  purin  bases  of  the  nucleins  into  uric  acid. 

The  uric  acid,  in  so  far  as  it  is  produced  from  nuclein  bases,  is  in  part  de- 
rived from  the  nucleins  of  the  destroyed  cells  of  the  body  and  in  part  from  the 
nucleins  or  free  purin  bases  introduced  with  the  food.  It  is  therefore  pos- 
sible to  admit  with  Buriax  and  Schur  3  of  a  double  origin  for  the  uric 
acid  as  well  as  the  urinary  purine  (all  purin  bodies  of  the  urine  with  the  excep- 
tion o'  the  uric  acid),  namely,  an  endogenous  and  an  exogenous  origin. 
Buriax  and  Schur  attempted  to  determine  the  quantity  of  endogenous 
urinary  purins  by  feeding  with  sufficient  food,  but  as  free  as  possible  from 
purin  bodies,  and  they  found  that  this  quantity  was  constant  for  even- 
individual,  while  it  was  variable  for  different  persons.  Other  investigators, 
such  as  Schreiber  and  Waldvogel  and  Loewi,4  have  arrived  at  somewhat 
different  results,  or  they  draw  different  deductions  from  their  observations; 
still  they  do  not  change  it  essentially,  namely,  that  the  uric  acid  originating 
from  the  nucleins  is  partly  endogenous  and  partly  exogenous. 

In  man  and  other  mammalia,  the  greatest  amount  if  not  all  of  the 
uric  acid  originates  from  the  nucleins  or  their  purin  bases.  In  birds  the 
condition  is  different,  v.  Mach  5  has  shown  that  in  these  animals  a  part  of 
the  uric  acid  maybe  formed  from  the  purin  bodies.  The  chief  quantity 
of  uric  acid,  however,  is  undoubtedly  formed  in  birds  by  synthesis. 

The  formation  of  uric  acid  in  birds  is  increased  by  the  administration 
of  ammonium  salts  (v.  Schroder)  and  urea  acts  in  a  similar  manner  in 
these  animals  (Meyer  and  Jaffe).  Mixkowski  observed  in  geese  with 
extirpated  livers  a  very  significant  decrease  in  the  elimination  of  uric  acid, 
while  the  elimination  of  ammonia  was  increased  to  a  corresponding  desrree. 
This  indicates  a  participation  of  ammonia  in  the  formation  of  uric  acid  in 


1  As  it  is  not  within  the  scope  of  this  book  to  enter  into  a  discussion  of  the  numerous 
researches  on  this  subject,  we  will  refer  to  Wiener,  "Die  Harnsiiure,"  Ergebnisse  der 
Physiol.,  1,  Abt.  I,  1902. 

2Ber.  d.  d.  Chem.  Gesellsch.,  36. 

s  Pfluger's  Arch.,  80,  87,  and  94. 

*  Schreiber  and  Waldvogel,  Arch.  f.  exp.  Path.  u.  Pharm.,  42;  O.  Loewi,  ibid.,  44 
and  45. 

J/bui.,24. 


4SS  URINE. 

the  organism  of  birds ;  and  as  Minkowski  has  also  found  after  the  extirpa- 
tion of  the  liver  that  considerable  amounts  of  lactic  acid  occur  in  the  urine, 
it  is  probable  that  the  uric  acid  in  birds  is  produced  in  the  liver  by 
synthesis,  perhaps  from  lactic  acid  and  ammonia;  although  as  Salaskin" 
and  Zaleski  and  Lang  have  shown  after  the  extirpation  of  the  liver 
primarily  an  increase  in  the  formation  of  lactic  acid  occurs  and  this  causes 
an  increase  in  the  elimination  of  ammonia  (neutralization  ammonia).  The 
direct  proof  for  the  uric-acid  formation  from  ammonia  and  lactic  acid  in 
the  liver  of  birds  has  been  given  by  Kowalewski  and  Salaskin  *  by  means 
of  blood-transfusion  experiments  on  geese  with  extirpated  livers.  They 
observed  a  relatively  abundant  formation  of  uric  acid  after  the  addition 
of  ammonium  lactate  and  to  a  still  greater  extent  after  arginine.  They  not 
only  consider  ammonium  lactate,  but  also  amino  acids  as  substances  from 
which  the  uric  acid  can  be  produced  in  the  liver  by  synthesis.  Of  these 
leucin,  glycocoll,  and  aspartic  acid  increase  the  elimination  of  uric  acid  in 
birds  (v.  Knieriem  2),  but  whether  they  are  first  decomposed  with  the 
splitting  off  of  ammonia  is  still  unknown. 

The  possibility  of  a  formation  of  uric  acid  from  lactic  acid  has  been 
shown  in  another  manner  by  Wiener,3  namely,  by  feeding  birds  with  urea 
and  lactic  acid  and  different  non-nitrogenous  substances,  oxy,  ketonic,  and 
dibasic  acids  of  the  aliphatic  series.  The  dibasic  acids,  with  a  chain  of  3 
carbon  atoms  or  their  ureides,  showed  themselves  most  active  as  uric-acid 
formers,  and  Wiener  is  therefore  of  the  opinion  that  the  active  substances 
must  first  be  converted  into  dibasic  acids.  By  the  attachment  of  a  urea 
residue  the  corresponding  ureid  is  produced,  according  to  Wiener,  and 
from  this  the  uric  acid  is  derived  by  the  attachment  of  a  second  urea  residue. 

Among  the  substances  tested,  only  tartronic  acid  and  its  ureid,  dialuric 
acid,  have  shown  themselves  active  in  the  experiments  with  the  iso- 
lated organs,  and  Wiener  therefore  also  considers  that  the  other  acids 
must  be  first  converted  into  tartronic  acid  by  oxidation  or  reduction. 
From  lactic  acid,  CH3.CH(OH).COOH,  we  first  obtain  tartronic  acid, 
COOH.CH(OH).COOH,  which  by  the  attachment  of  a  urea  residue  forms 

•\ttt pr\ 

dialuric  acid,  CO<Ntt /-,q>CHOH,  and  from  this  by  the  attachment  of  a 

second  urea  residue  uric  acid  is  formed. 

We  cannot  give  any  positive  answer  as  to  the  question  whether  uric  acid 


1  v.  Schroder,  Zeitschr.  f.  physiol.  Chem.,  2;  Meyer  and  JafTe,  Ber.  d.  d.  Chem. 
Gesellsch.,  10;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21  and  31;  Salaskin  and 
Zaleski,  Zeitschr.  f.  physiol.  Chem.,  29;  Lang,  ibid.,  32;  Kowalewski  and  Salaskin, 
ibid.,  33. 

2  Zeitschr.  f.  Biologie,  13. 

3  Hofmeister's  Beitriige,  2.  See  also  Arch.  f.  exp.  Path.  u.  Pharm.,  42,  and  Ergeb- 
tiisse  d.  Physiol.,  1,  Abt.  I,  1902. 


FORMATION  OF   URIC  ACID.  489 

is  formed  by  synthesis  also  in  man  and  other  mammalia.     WlENEB  has 
in   part  reported  experiments  which  seem   to   indicate  a  synthetic  uric-f 
acid  formation  in  the  isolated  mammalian  liver,  and  be  has  also  obtained 
an  increase  in  the  uric-acid   elimination,  although   only  a  Blight  one,  after 
feeding  lactic  acid  and  dialuric  acid  to  man. 

The  liver  seems  to  be  the  organ  in  birds  where  the  synthetical  forma- 
tion of  uric  acid  occurs,  and  the  fact  that  it  was  possible  for  Minkowski  « to 
arrest  the  uric-acid  formation  by  the  extirpation  of  the  liver  apparently 
shows  that  the  liver  Is  the  only  organ  taking  part  in  this  synthesis.  If  a 
synthesis  of  uric  acid  also  occurs  in  man  and  other  mammalia  we  must  con- 
sider the  liver  as  at  least  one  of  the  organs  taking  part  in  the  work,  as 
shown  by  Wiknkh's  investigations.  The  formation  of  uric  acid  from  nu- 
cleins  by  oxidation  has  often  been  connected  with  the  functions  of  the 
spleen,  but  there  are  no  grounds  for  such  a  view.  Mendel  and  Jackson 
have  indeed  shown  that  in  splenectomized  dogs  the  elimination  of 
uric  acid  was  considerably  increased  after  feeding  lymph-glands  or 
pancreas.  The  spleen  can  therefore  not  be  the  most  essential  organ  in 
this  type  of  uric-acid  formation.  The  experiments  made  with  liver  ex- 
tracts (Spitzer  and  Wiener  2),  in  which  it  was  possible  to  convert  purin 
bases  into  uric  acid,  showed  that  the  liver  has  also  the  same  power  of 
forming  uric  acid  as  the  spleen  has,  and  it  is  most  likely  that  the  uric 
acid  is  formed  in  the  different  organs,  in  which  a  destruction  of  nuclein 
tissue  takes  place. 

Uric  acid  when  introduced  into  the  mammalian  organism  is,  as  first 
shown  by  Wohler  and  Frerichs  for  the  dog  and  later  substantiated 
by  several  experimenters,3  in  great  part  destroyed  and  more  or  less  com- 
pletely changed  into  urea.  This  does  not  seem  to  be  the  same  for  all  ani- 
mals. In  rabbits,  according  to  Wiener,  the  uric  acid  is  destroyed  with 
the  formation  of  glycocoll  as  an  intermediate  step.  The  statements  are 
very  contradictory  with  carnivora.  According  to  an  older  view,  which  has 
received  support  by  the  recent  investigations  of  Salkowbki,  a  part  of 
the  uric  acid  introduced  into  dogs  is  eliminated  as  allantoin,  which  is  also 
true  according  to  Mendel  and  Brown  for  cats.  The  correctness  of  this 
view  is  denied  by  Wiener,  Pohl  and  Poduschka,4  still  we  cannot  con- 


>L.  c. 

:  Wiener,  1.  c. ;  Mendel  and  Jackson,  Amer.  Journ.  of  Physiol.,  4;  Spitzer,  Pfliiger's 
Arch.,  76. 

3  Wohler  and  Frerichs,  Annal.  d.  Chem.  u.  Pharm.,  65.  See  also  Wiener,  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  I. 

*  Wiener,  Arch.  f.  exp.  Path.  u.  Pharm.,  40  and  42.  and  Ergebnisse  der  Physiologie, 
1,  Abt.  I;  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  48;  Poduschka,  ibid.,  44;  Salkowski, 
Z.iKchr.  f.  physiol.  Chem.,  35,  and  Ber.  d.  d.  Chem.  Gesellsch.,  9;  Mendel  and  Brown, 
:.  Journ.  of  Physiol.,  3. 


490  URINE. 

sider  it  as  disproved.     The  possibility  of  a  uric-acid  formation  with  allan- 
toic as  an  intermediate  step  is  even  more  probable  in  man. 

The  destruction  of  uric  acid  seems  to  be  possible  in  several  organs,  and 
in  this  regard  also  different  animals  show  some  variation.  According  to 
the  researches  of  Chassevant  and  Richet,  Ascoli,  Jacoby  and  Wiener,1 
the  liver  of  the  dog  has  a  pronounced  power  of  destroying  uric  acid  and 
the  liver  of  the  pig  has  a  similar  power,  while  in  the  ox-liver  a  uric-acid- 
forming  activity  was  shown.  Other  organs  which  show  a  uric-acid-de- 
structive action  are,  according  to  Wiener,  the  kidneys,  which  action  in 
dogs  is  very  weak,  and  the  muscles. 

From  this  power  of  the  different  organs  of  destroying  uric  acid  it  follows 
that  the  quantity  of  uric  acid  eliminated  is  not  a  sure  indication  of  the 
amount  of  the  acid  formed.  We  must  admit,  therefore,  that  a  part  of  the 
uric  acid  formed  in  the  body  is  destroyed  in  a  similar  manner  to  that 
introduced  from  without.  Burian  and  Schur  2  have  indeed  suggested  a 
factor,  the  so-called  "integral  factor,"  with  which  the  quantity  of  uric  acid 
eliminated  in  the  twenty-four  hours  must  be  multiplied  in  order  to  find 
the  quantity  of  uric  acid  formed  during  this  time.  According  to  them, 
carnivora  eliminate  about  ^5— to  °f  the  uric  acid  introduced  into  the  cir- 
culation, rabbits  about  £,  and  man  \. 

Properties  and  Reactions  of  Uric  Acid.  Pure  uric  acid  is  a  white,  odor- 
less, and  tasteless  powder  consisting  of  very  small  rhombic  prisms  or  plates. 
Impure  uric  acid  is  easily  obtained  as  somewhat  larger,  colored  crystals. 

In  quick  crystallization,  small,  thin,  four-sided,  apparently  colorless, 
rhombic  prisms  are  formed,  which  can  be  seen  only  by  the  aid  of  the  micro- 
scope, and  these  sometimes  appear  as  spools  because  of  the  rounding  of 
their  obtuse  angles.  The  plates  are  sometimes  six-sided,  irregularly  devel- 
oped; in  other  cases  they  are  rectangular  with  partly  straight  and  partly 
jagged  sides ;  and  in  other  cases  they  show  still  more  irregular  forms,  the 
so-called  dumb-bells,  etc.  In  slow  crystallization,  as  when  the  urine  de- 
posits a  sediment  or  when  treated  with  acid,  large,  invariably  colored  crys- 
tals separate.  Examined  with  the  microscope  these  crystals  always  appear 
yellow  or  yellowish-brown  in  color.  The  most  ordinary  type  is  the  whet- 
stone shape,  formed  by  the  rounding  off  of  the  obtuse  angles  of  the  rhombic 
plate.  The  whetstones  are  generally  connected  together,  two  or  more 
crossing  each  other.  Besides  these  forms,  rosettes  of  prismatic  crystals, 
irregular  crosses,  brown-colored  rough  masses  of  destroyed  needles  and 
prisms  occur,  as  well  as  other  forms. 

Uric  acid  is  insoluble  in  alcohol  and  ether ;  it  is  rather  easily  soluble  in 
boiling  glycerine,  very  difficultly  soluble  in  cold  water,  in  39,480  parts  at 

'Chassevant  et  Richet,  Compt.  rend.  soc.  biolog.,  49;  Ascoli,  Pfliiger'a  Arch.,  72; 
Jacoby,  Virchow's  Arch.,  157;   Wiener,  Arch.  £.  exp.  Path.  u.  Pharm.,  42. 
'Pfluger's  Arch.,  87. 


PROPERTIES  OF  URIC  ACID.  401 

1S°C*  (His  and  Paul).  At  this  temperature,  according  to  them,  9.5  per 
cent  of  the  uric  acid  is  dissociated  in  the  saturated  solution.  Because  of 
the  reduction  in  the  dissociation  on  the  addition  of  Strong  acids  uric  acid 
is  soluble  with  difficulty  in  the  presence  of  mineral  acids.  It  Is  soluble 
in  a  warm  solution  of  sodium  diphosphate,  and  in  the  presence  of  an  excess 
of  uric  acid  monophosphate  and  acid  urate  are  produced.  The  ordi- 
nary view  is  that  sodium  diphosphate  forms  a  solvent  for  the  uric  acid  in 
the  urine,  but  according  to  Smale  the  monophosphate  has  only  a  slight  sol- 
vent action.  According  to  Rudel  l  urea  is  an  important  solvent,  but  this 
statement  has  not  been  confirmed  by  the  observations  of  His  and  Paul. 
Uric  acid  is  not  only  dissolved  by  alkalies  and  alkali  carbonates,  but  also  by 
several  organic  bases,  such  as  ethylamine  and  propylamine,  piperidin  and 
piperazin.  Uric  acid  dissolves  without  decomposing  in  concentrated 
sulphuric  acid.  It  is  completely  precipitated  from  the  urine  by  picric 
acid  (Jaffe  2).  Uric  acid  gives  a  chocolate-brown  precipitate  with  phospho- 
tungstic  acid  in  the  presence  of  hydrochloric  acid. 

Uric  acid  is  dibasic  and  correspondingly  forms  two  series  of  salts,  neu- 
tral and  acid.  Of  the  alkali  tirates  the  neutral  potassium  and  lithium  salts 
are  most  easily  soluble  and  the  ammonium  salt  dissolves  with  difficulty. 
The  acid-alkali  urates  are  very  insoluble  and  separate  as  a  sediment  {sedi- 
ment um  lateritium)  from  concentrated  urine  on  cooling.  The  salts  with 
alkaline  earths  are  very  insoluble. 

If  a  little  uric  acid  in  substance  is  treated  on  a  porcelain  dish  with  a 
few  drops  of  nitric  acid,  the  uric  acid  dissolves  on  warming  with  a  strong 
development  of  gas,  and  after  thoroughly  drying  on  the  water-bath  a 
beautiful  red  residue  is  obtained,  which  turns  a  purple-red  (ammonium 
purpurate  or  murexid  on  the  addition  of  a  little  ammonia.  If,  instead  of 
the  ammonia,  we  add  a  little  caustic  soda  (after  cooling),  the  color  becomes 
deeper  blue  or  bluish  violet.  This  color  disappears  quickly  on  warming,  differ- 
ing from  certain  xanthine  bodies.     This  reaction  is  called  the  murexid  test. 

If  uric  acid  is  converted  into  alloxan  by  the  careful  action  of  nitric  acid 
and  the  excess  of  acid  carefully  expelled,  on  treating  this  with  a  few  drops 
of  concentrated  sulphuric  acid  and  commercial  benzene  (containing  thio- 
phene),  a  beautiful  blue  coloration  is  obtained  (Deniges'  reaction  3). 

Uric  acid  does  not  reduce  an  alkaline  solution  of  bismuth,  while,  on  the 
contrary,  it  reduces  an  alkaline  cupric  hydrate  solution.  In  the  presence  of 
only  a  little  copper  salt  we  obtain  a  white  precipitate  consisting  of  cuprous 
urate.  In  the  presence  of  more  copper  salt  red  cuprous  oxide  separates. 
The  combination  of  uric  acid  with  cuprous  oxide  is  formed  when  copper 

1  His  and  Paul,  Zeitschr.  f.  physiol.  Chem.,  31;  Smale,  Centralbl.  f.  Physiol..  9 j 
Rudel,  Arch.  f.  exp.  Path.  u.  Pharm.,  30. 

2  Zeitschr.  f .  physiol.  Chem. ,  10- 

3  Journ.  de  Pharm.  et  de  Chim.,  18.     Cited  from  Italy's  Jahresber.,  18. 


492  URINE. 

salts  are  reduced  in  alkaline  solution  in  the  presence  of  urate  by  dextrftse  or 
bisulphite. 

If  a  solution  of  uric  acid  in  water  containing  alkali  carbonate  is  treated 
with  magnesium  mixture  and  then  a  silver-nitrate  solution  added,  a  gelati- 
nous precipitate  of  silver-magnesium  urate  is  formed.  If  a  drop  of  uric  acid 
dissolved  in  sodium  carbonate  is  placed  on  a  piece  of  filter-paper  which  has 
been  previously  treated  with  silver-nitrate  solution,  a  reduction  of  silver 
oxide  occurs  producing  a  brownish-black  or,  in  the  presence  of  only  0.002 
milligram  of  uric  acid,  a  yellow  spot  (Schiff's  test). 

The  precipitation  of  free  uric  acid  from  its  alkali  salts  by  means  of 
acids  can  be  more  or  less  prevented  by  the  presence  of  thymic  acid  or  nucleic 
acid  (Goto  x)  .  It  is  questionable  whether  this  is  of  any  physiological 
importance. 

Preparation  of  Uric  Acid  from  Urine.  Filtered  normal  urine  is  treated 
with  20-30  c.  c.  of  25  per  cent  hydrochloric  acid  for  each  liter  of  urine. 
After  forty-eight  hours  collect  the  crystals  and  purify  them  by  redissolving 
in  dilute  alkali,  decolorizing  with  animal  charcoal  and  reprecipitating  with 
hydrochloric  acid.  Large  quantities  of  uric  acid  are  easily  obtained  from 
the  excrements  of  serpents  by  boiling  them  with  dilute  caustic  potash  (5  per 
cent)  until  no  more  ammonia  is  developed.  A  current  of  carbon  dioxide 
is  passed  through  the  filtrate  until  it  barely  has  an  alkaline  reaction;  dis- 
solve the  separated  and  washed  acid  potassium  urate  in  caustic  potash,  and 
precipitate  the  uric  acid  in  the  filtrate  by  addition  of  an  excess  of  hydro- 
chloric acid. 

Quantitative  Estimation  of  Uric  Acid  in  the  Urine.  As  the  older 
method  suggested  by  Heintz,  even  after  recent  modifications,  gives 
inaccurate  results,  it  will  not  be  considered  here. 

Salkowski  and  Ludwig  's  2  method  consists  in  precipitating  by  silver 
nitrate  the  uric  acid  from  the  urine  previously  treated  with  magnesium 
mixture,  and  weighing  the  uric  acid  obtained  from  the  silver  precipitate. 
Uric-acid  determinations  by  this  method  are  often  performed  according  to 
the  suggestion  of  E.  Ludwig,  which  requires  the  following  solutions: 

1.  An  ammoniacal  silver-nitrate  solution,  which  contains  in  1  liter  26 
grams  of  silver  nitrate  and  a  quantity  of  ammonia  sufficient  to  completely  redissolve 
the  precipitate  produced  by  the  first  addition  of  ammonia.  2.  Magnesium  mixture. 
Dissolve  100  grams  of  crystallized  magnesium  chloride  in  water  and  add  enough 
ammonia  so  that  the  liquid  smells  strongly  of  it,  and  enough  ammonium  chloride 
to  dissolve  the  precipitate  and  dilute  the  solution  to  1  liter.  3.  Sodium-sulphide 
solution.  Dissolve  10  grams  of  caustic  soda  which  is  free  from  nitric  acid  and 
nitrous  acid  in  1  liter  of  water.  One  half  of  this  solution  is  completely  saturated 
with  sulphuretted  hydrogen  and  then  mixed  with  the  other  half. 

The  concentration  of  the  three  solutions  is  so  arranged  that  10  c.  c.  of 
each  is  sufficient  for  100  c.  c.  of  the  urine. 

1  Zeitschr.  f.  physiol.  Chem.,  30. 

2  Salkowski,  Virchow's  Arch.,  .">2,  Pfliiger's  Arch.,  5;  Salkowski,  Laboratory  Manual 
of  Physiol,  and  Path.  Chem.,  translated  by  Orndorff,  1904;  Ludwig,  Wien.  med. 
Jahrbuch,  18S4,  and  Zeitschr.  f.  anal.  Chem.,  24. 


ESTIMATION  OF  URIC  ACID.  493 

100-200  c.  c,  According  to  concentration,  of  the  filtered  urine  freed 
from  proteid  (by  boiling  after  the  addition  of  a  few  drops  <>f  acetic  acid)  is 
ed  into  a  beaker.  In  another  vessel  mix  10-20  c.  c.  of  the  silver  solu- 
tion with  10-L0  c.  c.  of  the  magnesium  mixture  and  add  ammonia,  and  when 
necessary  also  some  ammonium  chloride,  until  the  mixture  is  clear.  This 
solution  is  added  to  the  urine  while  stirring,  and  the  mixture  allowed  to 
stand  quietly  for  half  an  hour.  The  precipitate  is  collected  on  a  filter, 
washed  with  ammoniacaJ  water,  and  then  returned  to  the  same  beaker  by 
the  aid  of  a  glass  rod  and  a  wash-bottle,  without  destroying  the  filter. 
Now  heat  to  boiling  10-20  c.  c.  of  the  alkali-sulphide  solution,  which  has 
previously  been  diluted  with  an  equal  volume  of  water,  and  allow  this  solu- 
tion to  flow  through  the  above  filter  into  the  beaker  containing  the  silver 
precipitate;  wash  with  boiling  water,  and  wrarm  the  contents  of  the  beaker 
on  a  water-bath  for  a  tune,  stirring  constantly.  After  cooling  filter  into  a  por- 
celain dish,  wash  the  filter  with  boiling  water,  acidify  the  filtrate  with  hydro- 
chloric acid,  evaporate  it  to  about  15  c.  c,  add  a  few  drops  more  of  hydro- 
chloric acid,  and  allow  it  to  stand  for  twenty-four  hours.  The  uric  acid 
which  has  crystallized  is  collected  on  a  small  weighed  filter,  washed  with 
water,  alcohol,  ether,  and  carbon  disulphidc,  dried  at  100-110°  C,  and 
weighed.  For  each  10  c.  c.  of  watery  filtrate  we  must  add  0.00048  gram 
uric  acid  to  the  quantity  found  directly.  Instead  of  the  weighed  filter- 
paper  a  glass  tube  filled  with  glass-wool  as  described  in  other  handbooks 
may  be  substituted  (Ludwig).  Too  intense  or  continuous  heating  with 
the  alkali  sulphide  must  be  prevented,  otherwise  a  part  of  the  uric  acid 
may  be  decomposed. 

Salkowski  differs  from  this  procedure  by  precipitating  the  urine  first 
■until  a  magnesium  mixture  (50  c.  c.  to  200  c.  c.  urine) ,  filling  up  to  300  c.  c, 
and  filtering.  Of  the  filtrate,  200  c.  c.  is  precipitated  by  10-15  c.  c.  of  a 
3  per  cent  silver-nitrate  solution.  The  silver  precipitate  is  shaken  with  200- 
300  c.  c.  of  water  acidified  with  a  few  drops  of  hydrochloric  acid,  decomposed 
by  sulphuretted  hydrogen,  heated  to  boiling,  the  silver-sulphide  precipitate 
boiled  with  fresh  water,  filtered,  the  filtrate  concentrated  to  a  few  cubic 
centimeters,  treated  with  5-8  drops  of  hydrochloric  acid,  and  allowed  to 
stand  until  the  next  day. 

Hopkix's  method  is  based  on  the  fact  that  the  uric  acid  is  completely 
precipitated  from  the  urine  as  ammonium  urate  on  saturating  with  am- 
monium chloride.  The  uric  acid  can  either  be  weighed  after  being  set  free 
by  hydrochloric  acid  or  it  can  be  determined  in  several  ways,  by  titration 
with  potassium  permanganate  or  by  the  Kjeldahl  method.  Several  modi- 
fications of  this  method  have  been  worked  out  by  Folix,  Folix  and  Schaf- 
fer,  Worxer  and  Jolles.1  The  last  mentioned  convert  the  uric  acid 
into  urea  by  oxidation  with  potassium  permanganate  in  sulphuric-acid 
solution  and  then  determine  the  quantity  of  this  by  sodium  hypobromite. 
Of  these  methods  we  will  only  describe  that  suggested  by  Folix-Schafff.r. 

FoHn-Schaffer  Method.  Treat  300  c.  c.  urine  with  75  c.  c.  of  a  solution 
containing  500  grams  of  ammonium  sulphate,  5  grams  of  uranium  acetate,  and 
60  c.  c.  of  10  per  cent  acetic  acid,  and  filter  after  five  minutes.     This  removes 


1  Hopkins,  Journ.  of  Path,  and  Bact.,  1893.  and  Proceed.  Roy.  Soc,  52;  Folin, 
Zeitsehr.  f.  physiol.  Chem.,  24;  Folin  and  Schaffer,  ibid.,  32;  WOrner,  ibid.,  29;  Jolles, 
ibid.,  29,  and  Wien.  med.  Wochenschr. ,  1903. 


494  URINE. 

an  unknown  constituent  of  the  urine  (a  protein  substance?)  which  would 
otherwise  contaminate  the  uric  acid.  Take  125  c.  c.  of  the  nitrate  (corre- 
sponding to  100  c.  c.  of  the  urine)  and  add  5  c.  c.  of  concentrated  ammonia. 
After  twenty-four  hours  the  p  ecipitate  is  filtered  off  and  washed  free  from 
chlorine  on  the  filter  by  means  of  an  ammonium  sulphate  solution.  The 
precipitate  is  washed  off  the  filter  by  water  (to  al  100  c.  c.)  nto  a  flask, 
treated  with  15  c.  c.  of  concentrated  sulphuric  acid,  and  titrated  at 
60-63°  C.  with  N/20  potassium-permanganate  olution.  Ea  h  cubic  centi- 
meter of  this  solution  corresponds  to  3.75  milligrams  uric  acid.  Because  of 
the  solubility  of  the  ammonium  urate  a  correction  of  3  milligrams  must  be 
added  for  every  100  c.  c.  of  the  urine. 

In  regard  to  the  numerous  other  methods  for  estimating  uric  acid,  we 
must  refer  to  special  works  on  the  .  ubject,  and  especially  to  Huppert- 
Neubauer. 

Purin  Bases.  (Alloxuric  Bases.)  The  alloxuric  bases  (purin  bases) 
found  in  human  urine  are  xanthine,  guanine,  hypoxanthine,  adenine,  para- 
xanthine,  heteroxanthine,  episarkine,  epiguanine,  1-methylxanthine,  and  car- 
nine.  The  occurrence  of  guanine  and  carnine  (Pouchet)  'n,  according  to 
Kruger  and  Salomon/  not  positively  shown.  The  quantity  of  these  bodies 
in  the  urine  is  extremely  small  and  variable  in  different  individuals.  Fla- 
tow  and  Reitzenstein  2  found  15.6-45.1  milligrams  in  urine  voided  during 
twenty-four  hours.  The  quantity  of  alloxuric  bases  in  the  urine  is  increased 
regularly  after  feeding  with  nucleus  nucleins  or  food  rich  in  nucleins,  and 
after  free  destruction  of  leucocytes.  The  quantity  is  especially  increased 
in  leucaemia.  We  have  a  number  of  observations  on  the  elimination  of  thes  e 
bodies  in  different  diseases,  but  they  are  hardly  trustworthy,  on  account  of 
the  inaccuracy  of  the  methods  u?ed  in  the  determinations.  It  must  also 
be  remarked  that  the  three  alloxuric  bases,  heteroxanthine,  paraxanthine, 
and  1-methylxanthine,  which  form  the  chief  mass  of  the  alloxuric  bases  of 
the  urine,  are  derived,  according  to  the  investigations  of  Albanese,  Bond- 
zynski  and  Gottlieb,  E.  Fischer,  M.  Kruger  and  G.  Salomon  and 
Schmidt  3  from  the  theobromine,  caffeine,  and  theophylline  which  occur 
in  the  food.  With  the  purin  bases  we  must  also  differentiate  between 
those  of  endogenous  and  of  exogenous  origin.4    As  the  four  true  nuclein  bases 


1  Zeitschr.  f.  physiol.  Chem.,  24;  Pouchet,  "Contributions  a  la  connaissance  des 
matieres  extractives  de  l'urine. "  These  Paris,  1880.  Cited  from  Huppert-Neubauer, 
333  and  335. 

2  Deutsch.  med.  Wochenschr. ,  1897. 

3  Albanese,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Arch.  /.  exp.  Path.  u.  Pharm.,  35; 
Bondzynski  and  Gottlieb,  ibid.,  36,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  E. 
Fischer,  ibid  ,  30,  2405;  Kruger  and  Salomon,  Zeitschr  f.  physiol.  Chem.,  26;  Kruger 
and  Schmidt,  Ber.  d.  d.  chem.  Gesellsch.,  32,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  45. 

1  See  Burian  and  Schur,  foot-note  3,  page  487,  and  Kaufmann  and  Mohr,  Deutsch 
Arch.  f.  klin.  Med.,  74. 


PURIN   BASES.  495 

and  carnine  have  been  treated  in  Chapters  V  and  XI,  it  only  remains  to 
describe  the  special  urinary  purin  bodies. 

HN— CO 

Heteroxanthine,   CflHeN402  =  7-monomethylxanthine  =  OC     C.N.CH,,  was  first 

1      "     \CH 


HN— C.N4 

detected  in  the  urine  by  Salomon.  It  is  identical  with  the  monomethylxanthine 
which  passes  into  the  urine  after  feeding  with  theobromine  or  caffeine. 

Heteroxanthine  crystallizes  in  shining  needles  and  dissolves  with  difficulty 
in  cold  water  (1592  parte  at  18°  C).  It  is  readily  soluble  in  ammonia  and  alkalies. 
The  crystalline  sodium  salt  is  insoluble  in  strong  caustic  alkali  (33  per  cent)  and 
dissolves  with  difficulty  in  water.  The  chloride  crystallizes  beautifully,  is  rela- 
tively insoluble,  and  is  readily  decomposed  into  the  free  base  and  hydrochloric 
acid  by  water.  Heteroxanthine  is  precipitated  by  copper  sulphate  and  bisul- 
phite, mercuric  chloride,  basic  lead  acetate  and  ammonia,  and  by  silver  nitrate. 
The  silver  compound  dissolves  rather  easily  in  dilute,  warm  nitric  acid;  it  crystal- 
lizes in  small  rhombic  plates  or  prisms,  often  grown  together,  forming  charac- 
teristic crosses.  Heteroxanthine  does  not  give  the  xanthine  reaction,  but  does 
give  Weidel's  reaction,  especially  acco  ding  to  Fischer  (see  Chapter  V). 

CH3.N— CO 
I       I 

i-Methylxanthine,  C0H0N4O2  =         CO  C.NHX        ,  was  first  isolated  from  the 

I      II         >CH 
HN— C.N    / 

urine  and  studied  by  Kruger,  and  then  by  Kruger  and  Salomon.1  It  is  diffi- 
cultly soluble  in  cold  water,  but  readily  soluble  in  ammonia  and  caustic  soda, 
and  does  not  give  an  insoluble  sodium  combination.  It  is  readily  soluble  in 
dilute  acids,  and  it  crystallizes  from  its  acetic-acid  solution  in  thin,  generally 
hexagonal  plates.  The  chloride  is  decomposed  into  the  base  and  hydrochloric  acid 
by  water.  1-methylxanthine  gives  crystalline  double  salts  with  platinum  and 
gold.  It  is  not  precipitated  by  basic  lead  acetate,  and  when  pure  not  by  basic 
lead  acetate  and  ammonia.  With  ammonia  and  silver  nitrate  it  gives  a  gelatinous 
precipitate.  The  silver-nitrate  compound  crystallized  from  nitric  acid  forms 
rosettes  of  united  needles.  With  the  xanthine  test  with  nitric  acid  it  gives  an 
orange  coloration  on  the  addition  of  caustic  s  da.  It  gives  Weidel's  reaction 
(according  to  Fischer)  beautifully. 

CH3.N— CO 

Paraxanthine,     C7H8N402  =  1,7-dimethylxanthin   =         CO  C.N.CH, ,     urotheo- 

HN-C.N/CH 

bromine  (Thudichum),  was  first  isolated  from  the  urine  by  Thudichum  and  Salo- 
mon.2 It  crystallizes  beautifully  in  six-sided  plates  or  in  needles.  The  sodium 
combination  crystallizes  in  rectangular  plates  or  prisms  and,  like  the  hetero- 
xanthine-sodium  compound,  i  insoluble  in  33  per  cent  caustic-soda  solution. 
The  sodium  c  impound  separates  in  a  crystalline  state  on  neutralizing  its  solution 
in  water.  The  chloride  is  readily  soluble  and  is  not  decomposed  by  water.  The 
cbloroplatinate  crystalli  es  very  beautifully.  Mercuric  chloride  precipitates  it  only 
when  added  to  excess  and  after  a  long  time.  The  silver  nitrate  combination 
separates  as  white  silky  crystals  from  hot  nitric  acid  on  cooling.  It  gives  Weidel's 
reaction,  but  not  the  xanthine  test,  with  nitric  acid  and  alkali. 

1  Kruger,  Du  Bois-Reymond 's  Arch.,  1894;  Kruger  and  Salomon,  Zeitschr.  f. 
physiol.  Chem.,  24. 

2  Thudichum,  "Grundztige  d.  anal.  med.  klin.  Chemie  "  (Berlin,  1886);  Salomon, 
Du  Bois-Reymond 's  Arch.,  1882,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  16  and  18. 


496  URINE. 

Episarkine  is  the  name  given  by  Balke  to  a  purin  bodj7  occurring  in  human 
urine.  The  same  body  has  been  observed  by  Salomon  1  in  pigs '  and  dogs '  urine, 
as  well  as  in  urine  in  leucaemia.  Balke  gives  C4H8N30  as  the  probable  formula 
for  episarkine.  It  is  nearly  insoluble  in  cold  water,  dissolves  with  difficulty  in 
hot  water,  but  may  be  obtained  therefrom  as  long  fine  needles.  Episarkine  does 
not  give  the  xanthine  reaction  with  nitric  acid  nor  Weidel's  reaction.  With 
hydrochloric  acid  and  potassium  chlorate  it  gives  a  white  residue  which  turns 
violet  with  ammonia.  It  does  not  form  any  insoluble  sodium  compound.  The 
silver  combination  is  difficultly  soluble  in  nitric  acid.  Episarkine  is  possibly 
identical  with  epiguanine. 

HN— CO 
I       I 

Epiguanine,    C6H7N503  =  7-methylguanine  =  H2N.C     C.N.CH3,    was    first    pre- 

N~C.N/CH 
pared  from  the  urine  by  Kruger.2  It  is  crystalline  and  difficultly  soluble  in 
hot  water  or  ammonia.  It  crystallizes  from  a  hot  33  per  cent  caustic-soda  solu- 
tion on  cooling  in  broad  shining  crystals  and  dissolves  readily  in  hydrochloric  or 
sulphuric  acid.  It  gives  a  characteristic  chloroplatinate  crystallizing  in  six-sided 
prisms.  It  is  precipitated  by  neither  basic  lead  acetate  nor  by  basic  lead 
acetate  and  ammonia.  Silver  nitrate  and  ammonia  give  a  gelatinous  precipitate. 
It  responds  to  the  xanthine  test  with  nitric  acid  and  alkali.  According  to 
Eischer  it  acts  like  episarkine  with  Weidel's  test. 

In  preparing  alloxuric  bases  from  the  urine,  the  fluid  is  supersaturated  with 
ammonia  and  precipitated  by  a  silver-nitrate  solution.  The  precipitate  is  then 
decomposed  with  sulphuretted  hydrogen.  The  boiling-hot  filtrate  is  evaporated  to 
dryness  and  the  dried  residue  treated  with  3  per  cent  sulphuric  acid.  The  purin 
bases  are  dissolved,  while  the  uric  acid  remains  undissolved.  This  filtrate  is 
saturated  with  ammonia  and  precipitated  by  silver-nitrate  solution.  If  instead 
of  precipitating  with  silver  solution  we  desire  to  precipitate,  according  to  Kruger 
and  Wulff,  with  copper  suboxide,  the  urine  may  be  heated  to  boiling  and  imme- 
diately is  added,  successively,  100  c.  c.  of  a  50  per  cent  sodium-bisulphite  solution 
and  100  c.  c.  of  a  12  per  cent  copper-sulphate  solution  for  every  liter  of  urine. 
The  thoroughly  washed  preci  itate  i;  decomposed  with  hydrochloric  acid  and 
sulphuretted  hydrogen.  The  uric  acid  remains  in  great  part  on  the  filter.  Fur  Iher 
details  in  regard  to  the  treatment  of  the  solution  of  the  hydrochloric-acid  com- 
binations may  be  found  in  Kruger  and  Salomon.3 

Quantitative  Estimation  of  Alloxuric  Bases  according  to  Salkowski.4 
400  to  600  c.  c.  of  the  urine  free  from  proteid  is  first  precipitated  by 
magnesium  mixture  and  then  by  a  3  per  cent  silver-nitrate  solution  as 
described  on  page  493.  The  thoroughly  washed  silver  precipitate  is  de- 
composed by  sulphuretted  hydrogen  after  being  suspended  in  600-800 
c.  c.  of  water  with  the  addition  of  a  few  drops  of  hydrochloric  acid.  It  is 
heated  to  boiling  and  filtered  hot,  and  finally  evaporated  to  dryness  on 
the  water-bath.  The  residue  is  extracted  with  20-30  c.  c.  of  hot  3  per  cent 
sulphuric  acid  and  allowed  to  stand  twenty-four  hours ;  the  uric  acid  is  filtered 


1  Balke,  "Zur  Kenntniss  der  Xanthinkorper  "  (Inaug.-Diss.,  Leipzig,  1893);  Salo- 
mon, Zeitschr.  f.  physiol.  Chem.,  18. 

2Du  Bois-Reymond's  Arch.,  1894;  Kruger  and  Salomon,  Zeitschr.  f.  physiol. 
Chem.,  24  and  20. 

8  Zeitschr.  f.   physiol.  Chem.,  26,  and  also  Hoppe-Seyler-Thierf elder 's  Handbuch, 

7.  Aufl.,  154. 

♦Pfiuger's  Arch.,  69. 


OXALURIC  ACID.     OXALIC  ACID.  407 

off.  washed,  the  filtrate  made  ammoniacal,  and  the  xanthine  bodies  pre- 
cipitated again  by  silver  nitrate,  the  precipitate  collected  on  a  small,  chlo- 
rine-free filter,  washed  thoroughly,  dried,  carefully  incinerated,  the  ash 
dissolved  in  nitric  acid,  and  titrated  with  ammonium  sulphocyanide  accord- 
ing to  V0LHABD/s  method.  The  ammonium-sulphocvaiiide  solution  should 
contain  1.2-1.4  grams  per  liter  and  its  strength  should  be  determined  by  a 
silver-nitrate  solution:  1  part  silver  corresponds  to  0.277  gram  nitrogen  of 
alloxuric  bases  or  to  0.7381  gram  alloxuric  bases.  By  this  method  the 
uric-acid  and  alloxuric  bases  can  be  simultaneously  determined  in  the  same 
portion  of  urine.1 

M  \lfatti  2  determines  the  nitrogen  of  the  alloxuric  bases  in  the  hydrochloric 
acid  lilt  rate  from  the  separated  uric  acid.  This  filtrate  is  evaporated  with  mag- 
aesia  until  all  the  ammonia  has  been  expelled  and  the  residue  used  for  the 
Kjeldahl  determination. 

The  nitrogen  of  the  alloxuric  bases  is  also  determined  as  the  difference  between 
the  uric-acid  nitrogen  and  the  total  nitrogen  of  the  alloxuric  bodies  of  the  silver 
precipitate  (Camerer,  Arnstein3).  Salkowski  has  raised  the  objection  to 
;  his  procedure  that  it  is  not  possible  to  remove  all  the  ammonia  from  the  silver 
precipitate  by  washing.  According  to  Arnstein,4  this  can  readily  be  done  by 
boiling  the  precipitate  in  water  with  some  magnesia,  and  under  these  circum- 
stances this  method  is  quite  serviceable.  The  nitrogen  is  estimated  by  Kjel- 
DAHl's  method.  The  uric-acid  nitrogen  multiplied  by  3  gives  the  quantity  of 
uric  acid.  As  the  mixture  of  alloxuric  bases  in  the  urine  is  not  known,  the  quantity 
of  nitrogen  of  the  alloxuric  bases  is  always  calculated  as  a  certain  alloxuric  base, 
for  example,  xanthine  (Camerer),  and  the  quantity  so  found  used  as  a  measure 
for  the  alloxuric  bases. 

According  to  an  unpublished  method  of  Kruger  and  Schmid  (Hoppe-Seyler- 
Tuierfelder's  Handbuch,  7.  Aufl.,  435)  the  uric  acid  and  the  purin  bases  are 
precipitated  as  a  cuprous  compound  by  copper-sulphate  solution  and  sodium 
bisulphite.  The  precipitate  is  decomposed  in  sufficient  water  by  sodium  sulphide, 
and  the  uric  acid  precipitated  from  the  concentrated  filtrate  with  hydrochloric  acid, 
and  the  purin  bases  again  precipitated  from  this  filtrate  as  cuprous  or  silver  com- 
pounds. Finally,  the  nitrogen  in  the  uric-acid  part  and  the  part  containing 
the  mixture  of  purin  bases  is  estimated. 

We  cannot  discuss  the  other  methods  such  as  those  of  Deniges  and  Xiemi- 
lowicz,  and  the  method  suggested  bv  Hall  s  for  clinical  purposes. 

Oxaluric  Acid,  CJd4X204  =  (COX2H3).CO.COOH.  This  acid,  whose  relation 
to  uric  acid  and  urea  has  been  spoken  of  above,  does  not  always  occur  in  the  urine, 
and  then  only  in  traces  as  ammonium  salts.  This  salt  is  not  directly  precipi- 
tated by  CaCl2  and  XH„  but  after  boiling,  when  it  is  decomposed  into  urea  and 
oxalate.  In  preparing  oxaluric  acid  from  urine  the  latter  is  filter  d  th-  ough  animal 
charcoal.  The  oxalurate  retained  by  the  charcoal  may  be  obtained  by  boiling 
with  alcohol. 

POOTT 
Oxalic  Acid,  C2H204,  or  /W^rn  occurs  under  physiological  conditions, 

in  very  small  amounts  in  the  urine,  about  0.02  gram  in  twenty-four  hours 

1  In  regard  to  the  details  we  refer  the  reader  to  the  original  paper. 

*  Centralbl.  f.  innere  Med.,  1897. 

s  Camerer,  Zeitschr.  f.  Biologie,  26  and  28;  Arnstein,  Zeitschr.  f.  physiol.  Chem.,  23. 

4  Salkowski,  1.  c. ;   Arnstein,  Centralbl.  f.  d.  mod.  Wissensch.,  1898. 

5  Xiemilowicz,  Zeitschr.  f.  physiol.  Chem.,  35;  Gittelmacher-Wilenko,  ibid.,  36; 
Hall,  Wien.  klin.  Wochenschr. ,  16. 


49S  URINE. 

(Furbringer  l).  According  to  the  generally  accepted  view  it  exists  in 
the  urine  as  calcium  oxalate,  which  is  kept  in  solution  by  the  acid  phos- 
phates present.  Calcium  oxalate  is  a  frequent  constituent  of  urinary  sedi- 
ments and  occurs  also  in  certain  urinary  calculi. 

The  origin  of  the  oxalic  acid  in  the  urine  is  not  well  known.  Oxalic 
acid  when  administered  is  eliminated,  at  least  in  part,  by  the  urine  un- 
changed;2 and  as  many  vegetables  and  fruits,  such  as  cabbage,  spinach,, 
asparagus,  sorrel,  apples,  grapes,  etc.,  contain  oxalic  acid,  it  is  possible  that 
a  part  of  the  oxalic  acid  of  the  urine  originates  directly  from  the  food. 
That  oxalic  acid  may  be  formed  in  the  animal  body  as  metabolic  products 
from  proteids  or  fats  follows  from  the  observations  of  Mills  and  Luthje,* 
who  found  in  dogs  on  an  exclusively  meat  and  fat  diet,  as  also  in  starvation, 
that  oxalic  acid  was  eliminated  by  the  urine.  A  part  of  the  oxalic  acid 
may  also  be  due  to  a  greater  destruction  of  proteids  or,  as  found  by  Reale 
and  Boeri,  as  well  as  by  Terray,  a  greater  quantity  of  oxalic  acid  eliminated 
with  diminished  oxygen  supply  and  increased  proteid  destruction.  Pure 
proteid  does  not,  according  to  Salkowski,4  increase  the  quantity  of  oxalic 
acid  eliminated;  on  the  contrary,  after  meat  feeding  the  amount  of  this 
acid  is  increased,  due  in  part  to  the  meat  containing  oxalic  acid  (Sal- 
kowski). Gelatine  and  gelatine-yielding  tissues  seem  to  increase  the  ex- 
cretion of  oxalic  acid,  while  no  constant  increase  has  been  observed  after 
feeding  nucleins.5  The  production  of  oxalic  acid  due  to  an  incomplete 
combustion  of  the  carbohydrates  has  also  been  suggested.  The  work  of 
Hildebrandt  and  P.  Mayer  6  seems  to  indicate  this  under  abnormal 
conditions,  but  we  have  no  grounds  for  such  an  origin  for  oxalic  acid 
under  physiological  conditions.  The  same  is  true  for  the  formation  of 
oxalic  acid  by  oxidation  of  uric  acid  in  the  animal  body.7 

Oxalic  acid  is  best  detected  and  quantitatively  determined  according 
to  the  method  suggested  by  Salkowski:  Taking  out  the  oxalic  acid  from 
the  acidified  urine  by  means  of  ether  and  then  proceeding  as  follows  accord- 
ing to  Autenrieth  and  Barth:  8 

The  twenty-four-hour  urine  is  precipitated  by  CaCl2  and  ammonia 
in  excess.  After  18-20  hours  the  precipitate  is  collected  (the  filtrate 
must  be  clear)  and  dissolved  in  a  little  hydrochloric  acid  and  shaken  out 


'  Deutsch.  Arch.  f.  klin.  Med.,  18.     See  also  Dunlop,  Journ.  Path,  and  Bacterid  ,  3. 

2  In  regard  to  the  behavior  of  oxalic  acid  in  the  animal  body,  see  page  539. 

'Mills,  Virchow's  Arch.,  99;   Liithje,  Zeitschr.  f.  klin.  Med.,  35. 

4  Reale  and  Boeri,  Wien.  med.  Wochenschr.,  1895;  Terray,  Pfliiger's  Arch.,  65; 
Salkowski,  Berl.  klin.  Wochenschr.,  1900. 

*  See  Stradomsky,  Virchow's  Arch.,  163;  Mohr  and  Salomon,  Deutsch.  Arch.  f. 
klin.  Med.,  70;   Salkowski,  1.  c. 

8  Hildebrandt,  Zeitschr.  f.  physiol.  Chem.,  35;  P.  Mayer,  Zeitschr.  f.  klin.  Med.,  47- 

7  See  Wiener,  Ergebnisse  der  Physiol.,  1,  Abt.  I. 

8  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  29;   Autenrieth  and  Barth,  ibid.,  35. 


ALLANTOIC.  499 

4-5  times  with  150-200  c.  c.  'ether  (containing  3  per  cent  absolute 
alcohol).  'Nk'  united  ethereal  extracts  are  filtered  through  a  dry  filter 
and  distilled  after  the  addition  of  about  5  c.  c.  of  water.  The  liquid,  it" 
necessary,  is  decolorized  with  animal  charcoal  and  precipitated  with  CaCl3 
and  ammonia,  made  acid  after  a  certain  time  with  acetic  acid,  and  finally  the 
oxalate  is  collected,  washed,  burned,  to  CaO,  and  weighed. 

.NH.CH.HN. 
Allantoin  (glyoxyldiureid),  C4H6N403=OC<f  /CO,  occurs 

\NH.CO  H2.V 

in  the  urine  of  children  within  the  first  eight  days  after  birth,  and  in 
very  small  amounts  also  in  the  urine  of  adults  (Gtjsserow,  Ziegler  and 
Hermann).  It  is  found  in  rather  abundant  quantities  in  the  urine  of 
pregnant  women  (Gtjsserow).  Allantoin  has  also  been  found  in  the 
urine  of  sucking  calves  Wohler)  and  sometimes  in  the  urine  of  other 
animals  (MeHSNEr).  It  is  also  found  in  the  amniotic  fluid  and,  as  first 
shown  by  Vauquelin  and  Lassaigne,1  in  the  allantoic  fluid  of  the  cow 
(hence  the  name).  Allantoin  is  formed,  as  above  stated,  by  the  oxidation 
of  uric  acid  outside  of  th  animal  body,  hence  a  similar  formation  of  allan- 
toin is  admitted  in  the  animal  organism  (see  page  490).  According  to 
Poduschka  and  Minkowski,2  allantoin  introduced  into  dogs  appears 
almost  entirely  in  the  urine,  while  in  man  only  a  small  portion  of  the 
ingested  substance  is  eliminated  by  the  kidneys.  In  carnivora  the  excre- 
tion of  allantoin  is  considerably  inc  eased  according  to  Minkowski,  Cohn, 
Salkowski,  Mkndel  and  Brown3  after  feeding  thymus  or  pancreas.  A 
strong  allantoin  excretion  is  also  found  in  dogs  after  poisoning  with 
hydrazine  (Borissow),  hydroxylamine,  semicarbazide,  and  aminoguani- 
dine  (Pohl4).  He  also  obtained  allantoin  in  the  autolysis  of  the  intes- 
tinal mucosa,  liver,  thymus,  spleen,  and  pancreas.  As  no  allantoin  exists 
in  the  organs  of  normal  starving  dogs,  and  as  Pohl  has  found  it  in  the 
liver  and  as  traces  also  in  the  other  organs  after  poisoning  with  hydra- 
zine, he  claims  that  the  allantoin  is  formed  in  the  nuclein  destruction 
produced  by  the  death  of  the  cell-nuclei. 

Allantoin  is  a  colorless  substance  often  crystallizing  in  prisms,  difficultly 
soluble  in  cold  water,  easily  soluble  in  boiling  water,  and  also  in  warm 
alcohol,  but  not  soluble  in  cold  alcohol  or  ether.  A  watery  allantoin  solu- 
tion gives  no  precipitate  with  silver  nitrate  alone,  but  by  the  careful  addi- 
tion of  ammonia  a  white  flocculent  precipitate  is  formed,  C4H5AgN403,  which 

1  Ziegler  and  Hermann,  see  Gusserow,  Arch.  f.  Gynakol,  3 — both  cited  from  Hupport- 
Neubauer,  Harn- Analyse,  10.  Aufl.,  377;  Wohler,  Annal.  d.  Chem.  u.  Pharm.,  70; 
Meissner,  Zeitschr.  f.  rat.  Med.  (3),  31;  Lassaigne,  Annal.  de  Chim.  et  Phys.,  17. 

2  Arch.  f.  exp.  Path.  u.  Pharm  ,  44;   Minkowski,  ibid.,  41. 

3  Minkowski,  1.  c,  and  Centralbl.  f.  innore  Med.,  1898;  Cohn,  Zeitschr.  f.  physiol. 
Chem.,  25;  Salkowski,  Centralbl.  f.  d.  med.  Wissensch.,  1898;  Mendel  and  Brown, 
Amer.  Journ.  of  Physiol.,  3. 

'Arch.  f.  exp.  Path.  u.  Pharm.,  46. 


500  URINE. 

is  soluble  in  an  excess  of  ammonia  and  "which  consists  after  a  certain  time 
of  very  small,  transparent  microscopic  globules.  The  dry  precipitate- 
contains  40.75  per  cent  silver.  A  watery  allantoin  solution  is  precipitated 
by  mercuric  nitrate.  On  continuous  boiling  allantoin  reduce?  Feeling's 
solution.  It  gives  Schiff's  furfurol  reaction  less  rapidly  and  less  intensely 
than  urea.     Allantoin  does  not  give  the  murexid  test. 

Allantoin  is  most  easily  prepared  by  the  oxidation  of  uric  acid  with 
lead  peroxide.  In  preparing  allantoin  from  urine,  proceed  according  to 
Loewy's1  method,  which  consists  of  the  following:  The  faintly  acidified 
urine  is  precipitated  with  a  mercurous-nitrate  solution,  the  filtrate  treated 
with  H2S,  and  the  new  filtrate  precipitated  by  magnesium  oxide  and  silver 
nitrate  after  the  removal  of  the  H,S.  The  precipitate  is  filtered  off  and 
washed  with  warm  water  and  decomposed  with  H2S,  and  the  filtrate  evap- 
orated to  dryness.  The  residue  is  extracted  with  hot  water  and  then 
the  solution  precipitated  with  mercuric  nitrate.  The  precipitate  is  collected 
and  decomposed  by  H2S.  From  the  evaporated  filtrate  the  allantoin 
crystallizes  out.  This  method  can  be  used  for  the  quantitative  determina- 
tion of  allantoin. 

OC.C6H, 
Hippuric  Acid  (  benzoyl-amino  acetic  acid)  ,  CuHQNOo = 

F  HN.CH2.COOH. 

This  acid  decomposes  into  benzoic  acid  and  glycocoll  on  boiling  with 
mineral  acids  or  alkalies,  and  also  by  the  putrefaction  of  the  urine. 
The  reverse  of  this  occurs  if  these  two  components  are  heated  in  a  sealed 
tube  according  to  the  following  equation:  CeH5COOH  +  NH2.CH2.COOH  = 
C6H5.CO.XH.CH2.COOH  +  H20.  This  acid  may  be  synthetically  pre- 
pared from  benzamide  and  monochlor  acetic  acid,  C6H5.CO.NH2  +  CH2CL 
COOH  =  CGH5.CO.NH.CH2.COOH  +  HCl,  and  in  various  other  ways,  but 
simplest  from  glycocoll  and  benzoyl  chloride  in  the  presence  of  alkali. 

Hippuric  acid  occurs  in  large  amounts  in  the  urine  of  herbivora,  but 
only  in  small  quantities  in  that  of  carnivora.  The  quantity  of  hippuric 
acid  eliminated  in  human  urine  on  a  mixed  diet  is  usually  less  than  1  gram 
per  day;  as  an  average  it  is  0.7  gram.  After  eating  freely  of  vegetables 
and  fruit,  especially  such  fruit  as  plums,  the  quantity  may  be  more  than 
2  grams.  Hippuric  acid  is  also  found  in  the  perspiration,  blood,  suprarenal 
capsule  of  oxen,  and  in  ichthyosis  scales.  Nothing  is  positively  known  in 
regard  to  the  quantity  of  hippuric  acid  in  the  urine  in  disease. 

The  Formation  of  Hippuric  Acid  in  the  Organism.  Benzoic  acid  and 
also  the  substituted  benzoic  acids  are  converted  into  hippuric  acid  and  sub- 
stituted hippuric  acids  within  the  body.  Moreover,  those  bodies  are  trans- 
formed into  hippuric  acid  which  by  oxidation  (toluene,  cinnamic  acid, 
hydrocinnamic  acid)  or  by  reduction  (quinic  acid)  are  converted  into  ben- 
zoic acid.     The  question  of  the  origin  of  hippuric  acid  is  therefore  connected 


1  Arch.  f.  exp.  Path.  u.  Pharm.,  44. 


FORMATION  OF  HIPP  URIC  ACID.  501 

•with  the  question  of  the  origin  of  benzoic  acid;  the  formation  of  the 
second  component,  glycocoll,  from  the  protein  substances  in  the  body  ia 
unquestionable. 

Hippuric  acid  is  found  in  the  urine  of  starving  dogs  (Salkowski),  also 
in  dog's  urine  after  a  diet  consisting  entirely  of  meat  (Meisbneb  and 
Shepard,  Salkowski,  and  others  1).  It  is  evident  that  the  benzoic  acid 
originates  in  these  cases  from  the  proteids,  and  it  is  generally  admitted  that 
it  is  produced  by  the  putrefaction  of  proteids  in  the  intestine.  Among  the 
products  of  the  putrefaction  of  proteid  outside  of  the  body  Salkowski  has 
found  phenylpropionic  acid,  C6H5.CH2.CH2.COOH,  which  is  oxidized  in 
the  organism  to  benzoic  acid  and  eliminated  as  hippuric  acid  after  combin- 
ing with  glycocoll.  Phenylpropionic  acid  seems  to  be  formed  from  the 
aminophenylpropionic  acid,  which  is  derived  from  several  protein  substances. 
The  supposition  that  the  phenylpropionic  acid  is  produced  from  tyrosin  by 
putrefaction  in  the  intestine  has  not  been  substantiated  by  the  researches  of 
Baumann,  Schotten,  and  Baas.2  The  importance  of  putrefaction  in  the 
intestine  in  producing  hippuric  acid  is  evident  from  the  fact  that  after 
thoroughly  disinfecting  the  intestine  of  dogs  with  calomel  the  hippuric  acid 
disappears  from  the  urine  (Baumaxx  3). 

The  large  quantity  of  hippuric  acid  present  in  the  urine  of  herbivora  is 
partly  explained  by  the  specially  active  processes  of  putrefaction  going  on 
in  the  intestine  of  these  animals,  but  it  is  especially  due  to  the  large 
quantity  of  substances  in  the  plant-food  from  which  benzoic  acid  can  be 
formed.  There  is  hardly  any  doubt  that  the  hippuric  acid  in  human  urine 
after  a  mixed  diet,  and  especially  after  a  diet  of  vegetables  and  fruits,  origi- 
nates in  part  from  the  aromatic  substances,  e.  g.,  quinic  acid. 

The  view  proposed  by  Weiss  and  others  that  a  parallelism  exists  between 
the  excretion  of  hippuric  acid  and  uric  acid  in  that  an  increase  in  the  first  is 
followed  by  a  diminution  in  the  second  and  that,  for  example,  quinic  acid  produces 
a  diminution  in  the  excretion  of  uric  acid  corresponding  to  the  increased  forma- 
tion of  hippuric  acid  (Weiss,  Lewtin),  cannot  be  considered  as  sufficiently  proved  * 
(Hupfer). 

The  kidneys  may  be  considered  in  dogs  as  special  organs  for  the  syn- 
thesis of  hippuric  acid  (Schmiedeberg  and  Buxge  5).  In  other  animals, 
as  in  rabbits,  the  formation  of  hippuric  acid  seems  to  take  place  in  other 

'Salkowski,  Ber.  d.  deutsch.  chem.  Gesellsch.,  11:  Meissner  and  Shepard,  Cnter- 
such.  liber  das  Entstehen  der  Hippursaure  im  thierischen  Organismus.    Hannover,  1SG6. 

2E.  and  H.  Salkowski,  Ber.  d.  deutsch.  chem.  Gesellsch.,  12;  Baumann,  Zeitschr. 
f.  physiol.  Chem.,  7;   Schotten,  ibid.,  8;   Baas,  ibid.,  11. 

3  Ibid.,  10,  131. 

4  AVeiss,  Zeitsch.  f.  physiol.  Chem.,  2."i.  27.  38;  Lewin,  Zeitschr.  f.  klin.  Med.  42; 
Hupfer,  Zeitschr.  f.  physiol.  Chem.,  37.  See  also  Wiener,  "Die  Harnsaure,"  Ergeb- 
nisse  der  Physiol.,  1,  Abt.  I. 

5  Arch.  f.  exp.  Path.  u.  Pharm.,  0;  also  Ar.  Hoffmann,  ibid.,  7,  and  Kochs,  Pfliiger's 
Arch.,  20;   Bashford  and  Cramer,  Zeitschr.  f.  physiol.  Chem.,  35 


502  URINE. 

organs,  such  as  the  liver  and  muscles.  The  synthesis  of  hippuric  acid  is 
therefore  not  exclusively  limited  to  any  special  organ,  though  perhaps  in 
some  species  of  animals  it  may  be  more  abundant  in  one  organ  than  in 
another. 

Properties  and  Reactions  of  Hippuric  Acid.  This  acid  crystallizes  in 
semi-transparent,  long,  four-sided,  milk-white,  rhombic  prisms  or  columns, 
or  in  needles  by  rapid  crystallization.  They  dissolve  in  600  parts  cold 
water,  but  more  easily  in  hot  water.  They  are  easily  soluble  in  alcohol, 
but  with  difficulty  in  ether.  The  acid  dissolves  more  easily  (about  12  times) 
in  acetic  ether  than  in  ethyl  ether.     Petroleum  ether  hai  no  effect  upon  them. 

On  heating  hippuric  acid  it  first  melts  at  187.5°  C.  to  an  oily  liquid 
which  crystallizes  on  cooling.  By  continuing  the  heat  it  decomposes,  pro- 
ducing a  red  mass  and  a  sublimate  of  benzoic  acid,  with  the  generation, 
first,  of  a  peculiar  pleasant  odor  of  hay  and  then  an  odor  of  hydrocyanic 
acid.  Hippuric  acid  is  easily  differentiated  from  benzoic  acid  by  this 
behavior,  also  by  its  crystalline  form  and  its  insolubility  in  petroleum 
ether.  Hippuric  acid  and  benzoic  acid  both  give  Lucre  's  reaction,  namely, 
they  generate  an  intense  odor  of  nitrobenzene  when  evaporated  to  dryness 
with  nitric  acid  and  when  the  residue  is  heated  with  sand  in  a  glass  tube. 
Hippuric  acid  forms  crystallizable  salts,  in  most  cases,  with  bases.  The 
combinations  with  alkalies  and  alkaline  earths  are  soluble  in  water  and 
alcohol.  The  silver,  copper,  and  lead  salts  are  soluble  with  difficulty  in 
water;  the  ferric  salt  is  insoluble. 

Hippuric  acid  is  best  prepared  from  the  fresh  urine  of  a  horse  or  cow. 
The  urine  is  boiled  a  few  minutes  with  an  excess  of  milk  of  lime.  The 
liquid  is  filtered  while  hot,  concentrated  and  then  cooled,  and  the  hippuric 
acid  precipitated  by  the  addition  of  an  excess  of  hydrochloric  acid.  The 
crystals  are  pressed,  dissolved  in  milk  of  lime  by  boiling,  and  treated  as 
above;  the  hippuric  acid  is  precipitated  again  from  the  concentrated  fil- 
trate by  hydrochloric  acid.  The  crystals  are  purified  by  recrystallization 
and  decolorized,  when  necessary,  by  animal  charcoal. 

The  quantitative  estimation  of  hippuric  acid  in  the  urine  may  be  per- 
formed by  the  following  method  (Bunge  and  Schmiedeberg  *)  :  The  urine 
is  first  made  faintly  alkaline  with  soda,  evaporated  nearly  to  dryness,  and 
the  residue  thoroughly  extracted  with  strong  alcohol.  After  the  evapora- 
tion of  the  alcohol  the  residue  is  dissolved  in  water,  the  solution  acidified 
with  sulphuric  acid,  and  completely  extracted  by  agitating  (at  least  five 
times)  with  fresh  portions  of  acetic  ether.  The  acetic  ether  is  then  re- 
peatedly washed  with  water,  which  is  removed  by  means  of  a  separatory 
funnel,  then  evaporated  at  a  medium  temperature  and  the  dry  residue 
treated  repeatedly  with  petrolmim-ether,  which  dissolves  the  benzoic  acid, 
oxyacids,  fat,  and  phenols,  while  the  hippuric  acid  remains  undissolved. 
This  residue  is  now  dissolved  in  a  little  warm  water  and  evaporated  at 
50-60°  C.  to  crystallization.     The  crystals  are  collected  on  a  small  weighed 

1  Arch.  f.  exp.  Path  u.  Pharm.,  6.  In  regard  to  other  methods,  such  as  Blumen- 
thal  as  well  as  Pfeiffer,  Bloch  and  Riecke.  see  Maly's  Jahresber.,  30  and  32. 


ETHEREAL  SULPHURIC  ACIDS.  503 

filter.  The  mother-liquor  is  repeatedly  shaken  with  acetic  ether.  This 
last  is  removed  and  evaporated;  the  residue  is  added  to  the  above  crystals 
on  the  filter,  dried  and  weighed. 

Phenaceturic  Acid,  C10HnNO3  =  CeH5.CH2.CO.NH.CH2.COOH.  This  acid,  which 
is  produced  in  the  animal  body  by  a  combination  of  glyeocoll  with  the  phenyl- 
acetic  acid,  C9H5.CH2.COOH,  formed  in  the  putrefaction  of  the  proteids,  has 
been  prepared  from  horse's  urine  by  Salkowski,1  but  it  probably  also  occurs 
in  human  urine. 

Benzoic  Acid,  C7H802  or  CeH5.COOH,  is  found  in  rabbit's  urine  and  sometimes, 
though  in  small  amounts,  in  dog's  urine  (Weyl  and  v.  Anrep).  According  to 
Jaaksveld  and  Stokvis  and  to  Kronecker  it  is  also  found  in  human  urine  in 
diseases  of  the  kidneys.  The  occurrence  of  benzoic  acid  in  the  urine  seems  to 
be  due  to  a  fermentative  decomposition  of  hippuric  acid.  Such  a  decomposition 
may  very  easily  occur  in  an  alkaline  urine  or  in  one  containing  proteid  (Van  de 
Vei.de  and  Stokvis).  In  certain  animals — pigs  and  dogs — the  kidneys,  accord- 
ing to  Schmiedeberg  and  Minkowski,2  contain  a  special  enzyme,  Schmiedeberg's 
histozym,  which  splits  the  hippuric  acid  with  the  separation  of  benzoic  acid. 

Ethereal  Sulphuric  Acids.  In  the  putrefaction  of  proteids  in  the  intes- 
tine, phenols,  whose  mother-substance  is  considered  to  be  tyrosin,  and  indol 
and  skatol  are  produced.  These  phenols  directly,  and  the  two  last-named 
bodies  after  they  have  been  oxidized  respectively  into  indoxyl  and  skatoxyl, 
pass  into  the  urine  as  ethereal  sulphuric  acids  after  uniting  with  sulphuric 
acid.  The  most  important  of  these  ethereal  acids  are  phenol-  and  cresol- 
sulphuric  acids — which  were  formerly  also  called  phenol-forming  substances 
— indoxyl-  and  skatoxyl-sulphuric  acids.  To  this  group  belong  also  the 
pyrocatechin-sulphuric  acid,  which  occurs  only  in  very  small  amounts  in 
human  urine,  and  hydroquinonc-sulphuric  acid,  which  appears  in  the  urine 
after  poisoning  with  phenol,  and  under  physiological  conditions  perhaps 
other  ethereal  acids  occur  which  h.we  not  been  isolated.  The  ethereal 
sulphuric  acids  of  the  urine  were  discovered  and  specially  studied  by 
Baumann.3  The  quantity  of  these  acids  in  human  urine  is  small,  while 
horse's  urine  contains  larger  quantities.  According  to  the  determinations 
of  v.  d.  Velden  the  quantity  of  ethereal  sulphuric  acid  in  human  urine  in 
the  twenty-four  hours  varies  between  0.094  and  0.620  gram.  The  rela- 
tionship of  the  sulphate-sulphuric  acid  A  to  the  conjugated  sulphuric  acid 
B  in  health  is  on  an  average  as  10  : 1.  It  undergoes  such  great  variations,  as 
found  by  Baumaxx  and  Herter,4  and  after  them  by  many  other  investi- 
gators, that  it  is  hardly  possible  to  consider  the  average  figures  as  normal. 
After  taking  phenol  and  certain  other  aromatic  substances,  as  well  as  when 
putrefaction  within  the  organism  is  general,  the  elimination  of  ethereal  sul- 

1  Zeitschr.  f.  physiol.  Chem.,  9. 

7  Weyl  and  v.  Anrep,  Zeitschr.  f.  physiol.  Chem.,  4;  Jaarsveld  and  Stokvis,  Arch, 
f.  exp.  Path.  u.  Pharm.,  10;  Kronecker,  ibid.,  16;  Van  der  Velde  and  Stokvis,  ibid., 
17;  Schmiedeberg,  ibid.,  14,  379;  Minkowski,  >bid.,  17. 

8  Pfliiger's  Arch.,  12  and  13. 

4  v.  d.  Velden,  Virchow's  Arch.,  70;  Herter,  Zeitschr.  f.  physiol.  Chem.,  1. 


504  URINE. 

phuric  acid  is  greatly  increased.  On  the  contrary,  it  is  diminished  when  the 
putrefaction  in  the  intestine  is  reduced  or  prevented.  For  this  reason  it 
may  be  greatly  diminished  by  carbohydrates  and  exclusive  milk  diet.1  The 
intestinal  putrefaction  and  the  elimination  of  ethereal  sulphuric  acid  has 
also  been  diminished  in  certain  cases  by  certain  therapeutic  agents  which 
have  an  antiseptic  action;  still  the  statements  are  not  unanimous.2 

Great  importance  has  been  given  to  the  relationship  between  the  total 
sulphuric  acid  and  the  conjugated  sulphuric  acid,  or  between  the  conjugated 
sulphuric  acid  and  the  sulphate-sulphuric  acid,  in  the  study  of  the  intensity 
of  the  putrefaction  in  the  intestine  under  different  conditions.  Several 
investigators,  F.  Muller,  Salkowski,  and  v.  Noorden,3  consider  cor- 
rectly that  this  relationship  is  only  of  secondary  value,  and  that  it  is  more 
correct  to  consider  the  absolute  value.  It  must  be  remarked  that  the  abso- 
lute values  for  the  conjugated  sulphuric  acid  also  undergo  great  variation, 
so  that  it  is  at  present  impossible  to  give  the  upper  or  lower  limit  for  the 
normal  value. 

Phenol  -     and     p  -  Cresol  -  sulphuric     Acid,    C6H5 . 0.  S02 .  OH     and 

C6H4<pg    2'       .      These  acids  are  found  as  alkali  salts  in  human  urine, 

in  which  also  orthocresol  has  been  detected.  The  quantity  of  cresol-sulphuric 
acid  is  considerably  greater  than  phenol-sulphuric  acid  In  the  quantitative 
estimation  the  phenols  are  set  free  from  the  two  ethereal  acids  and  deter- 
mined together  as  tribromphenol.  The  quantity  of  phenols  which  are 
separated  from  the  ethereal-sulphuric  acids  of  the  urine  amounts  to  17-51 
milligrams  in  the  twenty-four  hours  (Munk).  The  methods  for  the  quantita- 
tive estimation  used  heretofore  give,  according  to  Rumpf,  as  well  as  Kossler 
and  Penny,4  such  inaccurate  results  that  new  determinations  are  very  desir- 
able. After  a  vegetable  diet  the  quantity  of  these  ethereal-sulphuric  acids  is 
greater  than  after  a  mixed  diet.  After  the  ingestion  of  carbolic  acid,  which 
is  in  great  part  converted  by  synthesis  within  the  organi  m  into  phenol- 
sulphuric  acid,  besides  also  pyrocatechin-  and  hydroqu'non-sulphuric 
acid,5  or  when  the  amount  of  sulphuric  acid  is  not  sufficient  to  combine 

1  See  Hirschler,  Zeitschr.  f.  physiol.  Chera.,  10;  Biernacki,  Deutsch.  Arch.  f.  klin. 
Med.,  49;  Rovighi,  Zeitschr.  f.  physiol.  Chem.,  16;  Winternitz,  ibid.,  and  Schmitz, 
ibid.,  17  and  19. 

2  See  Baumann  and  Morax,  Zeitschr.  f  physiol.  Chem.,  10;  Steiff,  Zeitschr.  f. 
klin.  Med.,  1G;  Rovighi,  1.  c. ;  Stern,  Zeitschr.  f.  Hygiene,  12;  and  Bartoschewitsch, 
Zeitschr.  f.  physiol.  Chem.,  17;   Mosse,  ibid.,  23. 

3  Midler,  Zeitschr.  f.  klin  Med.,  12;  v.  Noorden,  ibid.,  17;  Salkowski,  Zeitschr. 
f.  physiol.  Chem.,  12. 

*  Munk,  Pfliiger's  Arch.,  12;  Rumpf,  Zeitschr.  f.  physiol.  Chem.,  16;  Kossler  and 
Penny,  ibid.,  17. 

6  See  Baumann,  Pfluger's  Arch.,  12  and  13,  and  Baumann  and  Preusse,  Zeitschr. 
f,  physiol.  Chem.,  3,  156. 


PHENOL-  AND  CRESOL-SULPHURIC  ACIDS.  505 

with  the  phenol,  it  forms  phenyl-glucuronic:  acid.1  the  quantity  of  phenols 
and  ethereal-sulphuric  acids  id  the  urine  is  considerably  increased  at  the 
expense  of  the  sulphate-sulphuric  acid. 

An  increased  elimination  of  phenol-sulphuric  acids  occurs  in  active 
putrefaction  in  the  intestine  with  stoppage  of  the  contents  of  the  intestine, 
as  in  ileus,  diffused  peritoniti  with  atony  of  the  intestine,  or  tuberculous 
enteritis,  but  not  in  simple  obstruction.  The  elimination  is  also  increased 
by  the  absorption  of  the  product  of  putrefaction  from  purulent  wounds  or 
abscesses.  An  increased  elimination  of  phenol  has  been  observed  in  a  few 
other  cases  of  diseased  conditions  of  the  body.2 

The  alkali  salts  of  phenol-  and  cresol-sulphuric  acids  crystallize  in  white 
plates,  similar  to  mother-of-pearl,  which  are  rather  freely  soluble  in  water. 
They  are  soluble  in  boiling  alcohol,  but  only  slightly  soluble  in  cold.  On 
boiling  with  dilute  mineral  acids  they  are  decomposed  into  sulphuric  acid 
and  the  corresponding  phenol. 

Phenol-sulphuric  acids  have  been  synthetically  prepared  by  Baumann 
from  potassium  pyrosulphate  and  phenol-  or  p-cresol-potassium.  For  the 
method  of  their  preparation  from  urine,  which  is  rather  complicated,  and 
also  for  the  known  phenol  reactions,  the  reader  is  referred  to  other  text- 
books. The  quantitative  estimation  of  these  ethereal-sulphuric  acids  was 
usually  performed  by  weighing  the  phenol  which  was  separated  from  the 
urine  as  tribromphenol.  At  the  present  time  the  following  method  is 
employed: 

Kossler  and  Penny's  method  with  Neuberg's3  modification.  The 
liquid  containing  phenol  is  treated  with  N/10  caustic  soda  until  strongly 
alkaline,  warmed  on  the  water-bath  in  a  flask  with  a  glass  stopper,  and 
then  treated  with  an  excess  of  N/10  iodine  solution,  the  quantity  being 
exactly  measured.  Sodium  iodide  is  first  formed  and  then  sodium  hypo- 
iodite,  which  latter  forms  tri-iodophenol  with  the  phenol  according  to  the 
following  equation : 

C6H5OH  +  3NaIO  -  Cr,H2I3.OH  +  3NaOH. 

On  cooling  acidify  with  sulphuric  acid  and  determine  by  titration  with 
N/10  sodium  thiosulphate  solution  the  excess  of  iodine.  This  process 
is  also  available  for  the  estimation  of  paracresol.  Each  cubic  centimeter 
of  the  iodine  solution  used  is  equivalent  to  1.5670  milligrams  of  phenol  or 
1.8018  milligrams  of  cresol.  As  the  determination  does  not  give  any  idea 
as  to  the  variable  proportions  of  the  two  phenols,  the  quantity  of  iodine 
used  must  be  calculated  as  one  or  the  other  of  the  two  phenols.  Before 
such  a  determination  is  carried  out  the  concentrated  urine  is  first  distilled 
after  acidification  with  sulphuric  acid  and  the  distillate  purified  by  pre- 
cipitation with  lead  and  distilled  again  (Netjberg).  For  details,  see 
Neuuerg,  1.  c,  and  Hoppe-Seyler-Thierfelder's  Handbuch,  7.  Aufl. 


1  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  14. 

2  See  G.    Hoppe-Seyler,   Zeitschr.    f.    physiol.    Chem.,   12.     This   contains   also  all 
references  to  the  literature  on  this  subject.     Fedeli,  Moleschott'a  Untersuch.,  15. 

3  Kossler  and  Penny,  Zeitschr.  f.  physiol.  Chem.,  17;   Xeuberg,  ibid.,  27. 


506  URINE. 

The  methods  for  the  separate  determination  of  the  conjugated  sulphuric 
acid  and  the  sulphate-sulphuric  acid  will  be  spoken  of  later  in  connection 
with  the  determination  of  the  sulphuric  acid  of  the  urine. 

Pyrocatechin-sulphuric  Acid.  This  acid  was  first  found  in  horse's  urine  in 
rather  large  quantities  by  Baumann.  It  occurs  in  human  urine  only  in  the 
very  smallest  amounts,  and  perhaps  not  constantly,  but  it  is  present 
abundantly  in  the  urine  after  taking  phenol,  pyrocatechin,  or  protocatechuic 
acid. 

With  an  exclusive  meat  diet  this  acid  does  not  occur  in  the  urine,  and  it 
therefore  must  originate  from  vegetable  food.  It  probably  originates  from  the 
protocatechuic  acid,  which,  according  to  Preusse,  passes  in  part  into  the  urine 
as  pyrocatechin-sulphuric  acid.  This  acid  may  also  perhaps  depend  on  the 
oxidation  of  phenol  within  the  organism  (Baumann  and  Preusse  1). 

Pyrocatechin,  or  o-dioxybenzene,  C6H4(OH)2,  was  first  observed  in  the  urine 
of  a  child  (Ebstein  and  J.  Muller).  The  reducing  body  alcapton,  first  found 
by  Bodeker  2  in  human  urine  and  which  was  considered  for  a  long  time  as  ident- 
ical with  pyrocatechin,  is  in  most  cases  probably  homogentisic  acid  or  uroleucic 
acid  (see  below). 

Pyrocatechin  crystallizes  in  prisms  which  are  soluble  in  alcohol,  ether,  and 
water.  It  melts  at  10?-104°  C,  and  sublimes  in  shining  plates.  The  watery 
solution  becomes  green,  brown,  and  ultimately  black  in  the  presence  of  alkali  and 
the  oxygen  of  the  air.  If  very  dilute  ferric  chloride  is  treated  with  tartaric  acid 
and  then  made  alkaline  with  ammonia,  and  this  added  to  a  watery  solution 
of  pyrocatechin,  we  obtain  a  violet  or  cherry-red  liquid  which  becomes  green 
on  saturating  with  acetic  acid.  Py  ocatechin  is  precipitated  by  lead  acetate. 
It  reduces  an  ammoniacal  silver  solution  at  the  ordinary  temperature,  and  re- 
duces alkaline  copper-oxide  solutions  with  heat,  but  does  not  reduce  bismuth 
oxide. 

A  urine  containing  pyrocatechin,  if  exposed  to  the  air,  especially  when  alkaline, 
quickly  becomes  dark  and  reduces  alkaline  copper  solutions  when  heated.  In 
detecting  pyrocatechin  in  the  urine  it  is  concentrated  when  necessary,  filtered, 
boiled  with  the  addition  of  sulphuric  acid  to  remove  the  phenols,  and  repeatedly 
shaken  after  cooling  with  ether.  The  ether  is  distilled  from  the  several  ethereal 
extracts,  the  residue  neutralized  with  barium  carbonate  and  shaken  again  with 
ether.  The  pyrocatechin  which  remains  after  evaporating  the  ether  may  be 
purified  by  recrystallization  from  benzene. 

Hydroquinone,  or  p-dioxyben/ene,  C6H4(OH)2,  often  occurs  in  the  urine  after 
the  use  of  phenol  (Baumann  and  Preusse)  .  The  dark  color  which  certain  urines, 
so-called  ' '  carbolic  urines, ' '  assume  in  the  air  is  due  to  decomposition  products. 
Hydroquinone  does  not  occur  as  a  normal  constituent  of  urine,  but  after  the  ad- 
ministration of  hydroquinone,  according  to  Lewin,3  it  passes  into  the  urine  of 
rabbits  as  an  ethereal-sulphuric  acid,  being  a  decomposition  product  of 
arbutin. 

Hydroquinone  forms  rhombic  crystals  which  are  readily  soluble  in  water, 
alcohol,  and  ether.  It  melts  at  169°  C.  Like  pyrocatechin,  it  easily  reduces 
metallic  oxides.  It  acts  like  pyrocatechin  with  alkalies,  but  is  not  precipitated 
with  lead  acetate.  It  is  oxidized  into  quinone  by  ferric  chloride  and  other  oxidiz- 
ing agents,  and  quinone  can  be  detected  by  its  peculiar  odor.  Hydroquinone- 
sulphuric  acid  is  detected  in  the  urine  by  the  same  methods  as  pyrocatechin- 
sulphuric  acid. 

1  Baumann  and  Herter,  Zeitschr.  f.  physiol.  Chem.,  1;  Preusse,  ibid.,  2;  Baumann, 
ibid.,  3. 

2  Ebstein  and  Muller,  Virchow's  Arch.,  62;  Bodeker,  Zeitschr.  f.  rat.  Med.  (3),  7. 
8  Virchow's  Arch.,  92. 


JMjOXYL-SULPHURIC  ACID.  507 

CH 
Indoxyl-sulphuric  Acid, C8H7NSO,  =  HC      C— C.O.S02(OU ),  also  called 

HC      C     CH 

VV 
CH  Nil 

urine  ixdicax,  formerly  called  uroxaxthixe  (Heller),  occurs  as  an  alkali- 
salt  in  the  urine.  This  acid  is  the  mother-substance  of  a  great  part  of 
the  indigc  of  the  urine.  The  quantity  of  indigo  which  can  be  separated 
from  the  urine  is  considered  as  a  measure  of  the  quantity  of  indoxyl-sul- 
phuric  acid  (and  indoxyl-glucuronic  acid)  contained  in  the  urine.  This 
amount,  according  to  Jaffe,1  for  man  is  5-20  milligrams  per  twenty-four 
hours.  Horse's  urine  contains  about  twenty-five  times  as  much  indigo- 
forming  substance  as  human  urine. 

Indoxyl -sulphuric  acid  is  derived,  as  previously  mentioned  (page  335), 
from  indol,  which  is  first  oxidized  in  the  body  into  indoxyl  and  is  then 
coupled  with  sulphuric  acid.  After  subcutaneous  injection  of  indol  the  elimi- 
nation of  indican  is  considerably  increased  (Jaffe,  Baumaxx  and  Brieger). 
It  is  also  increased  by  the  introduction  of  orthonitrophenylpropiolic  acid 
in  the  animal  organism  (G.  Hoppe-Seyler  2).  Indol  is  formed  by  the 
putrefaction  of  proteids.  The  putrefaction  of  secretions  rich  in  proteid  in 
the  intestine  explains  also  the  occurrence  of  indican  in  the  urine  during 
starvation.  Gelatine,  on  the  contrary,  does  not  increase  the  elimination  of 
indican. 

An  abnormally  increased  elimination  of  indican  occurs  in  such  diseases  as 
obstruct  the  small  intestine,  causing  an  increased  putrefaction  and  thus  pro- 
ducing an  abundance  of  indol.  Such  an  increased  elimination  of  indican 
occurs  on  tying  the  small  intestine  of  a  dog,  but  not  the  large  intestine 
(Jaffe),  an  observation  which  has  been  confirmed  recently  by  Ellixgkr 
and  Prutz.3  They  removed  an  intestinal  loop  in  dogs  and  replaced  it 
in  a  reversed  position,  the  distal  end  of  the  loop  being  attached  to  the 
proximal  end  of  the  intestine,  and  in  this  manner,  by  the  inverted  peristalsis 
so  obtained  they  effected  a  disturbance  in  the  movement  of  the  intestinal 
contents.  It  was  shown  that  this  obstruction  in  the  small  intestine  caused 
an  increased  elimination  of  indican,  while  an  obstruction  in  the  large  intes- 
tine showed  no  such  action. 

The  putrefaction  of  proteids  in  other  organs  and  tissues  besides  the 
intestine  may  also  cause  an  increase  in  the  indican  of  the  urine.  Certain 
investigators,  Blumexthal,  Rosexfeld  and  Lewix,  claim  to  have  showo 

1  Pfliiger's  Arch.,  3. 

3  Jaffe.  Centralbl.  f.  d.  med.  Wissenseh.,  1872;  Baumann  and  Brieger,  Zeitschr.  t 
physiol.  Chem.,  I;  G.  Hoppe-Seyler,  ibid.,  7  and  8. 

3  Jaffe,  Virchow's  Arch.,  70;   Ellinger  and  Prutz,  Zeitschr.  f.  physiol.  Chem.,  38. 


508  URINE. 

that  an  increased  excretion  of  indican  can  be  brought  about  also  without 
putrefaction  by  an  increased  destruction  of  tissue  in  starvation  and  also  after 
phlorhizin  poisoning;  but  these  statements  are  actively  disputed  by  other 
investigators,  such  as  P.  Mayer,  Scholz,  and  Ellinger,1  and  are  still  un- 
settled. After  poisoning  with  oxalic  acid  Harnack  and  v.  Leyen  found  an 
increased  indican  elimination  and  Moraczewski  has  been  able  to  prove  a 
certain  parallelism  between  the  quantity  of  indican  and  the  quantity  of  oxalic 
acid  in  diabetes.  Scholz  2  obtained,  on  the  contrary,  no  increase  in  the 
excretion  of  indican  after  oxalic  acid.  An  increased  elimination  of  indican 
has  been  observed  in  many  diseases,3  and  in  these  cases  the  quantity  of  phenol 
eliminated  is  also  generally  increased.  A  urine  rich  in  phenol  is  not  always 
rich  in  indican. 

The  potassium  salt  of  indoxyl-sulphuric  acid,  which  was  prepared  pure 
by  Baumann  and  Brieger  from  the  urine  of  a  dog  fed  on  indol,  has  since 
been  prepared  synthetically  by  Baumann  and  Thesen,4  by  fusing  phenyl- 
glycin-orthocarbonic  acid  with  alkali  and  then  from  this  producing  the 
indoxylsulphate  by  means  of  potassium  pyrosulphate.  It  crystallizes  in 
colorless,  shining  plates  or  leaves  which  are  easily  soluble  in  water,  but  less 
readily  in  alcohol.  It  is  split  by  mineral  acids  into  sulphuric  acid  and 
indoxyl.  The  latter  without  access  of  air  passes  into  a  red  compound, 
indoxyl-red,  but  in  the  presence  of  oxidizing  reagents  is  converted  into 
indigo-blue:  2C8H7NO+2O  =  C16H10N2O2+2H2O.  The  detection  of  indican 
is  based  on  this  last  fact. 

For  the  rather  complicated  preparation  of  indoxyl-sulphuric  acid  as  the 
potassium  salt  from  urine  the  reader  is  referred  to  other  text-books.  For 
the  detection  of  indican  in  urine  in  ordinary  cases  the  following  method  of 
Jaffe-Obermayer,  which  also  serves  as  an  approximate  test  for  the  quan- 
tity of  indican,  is  sufficient. 

Jaffe-Obermayer  's  Indican  Test.  Jaffe  uses  chloride  of  lime  as  the 
oxidizing  agent,  while  Obermayer  employs  ferric  chloride.  Other  oxidizing 
agents  have  been  suggested,  such  as  potassium  permanganate,  potassium 
bichromate,  alkali  chlorate,  and  hydrogen  peroxide  (the  latter  suggested 
by  Porcher  and  Hervieux5).  With  Obermayer's  reagent  the  test  is 
performed  as  follows: 

1  Blumenthal,  Arch.  f.  (Anat.  u.)  Physiol.,  1901,  Suppl.,  and  1902,  with  Rosenfeld, 
Charit6  annalen,  27;  Lewin,  Hofmeister's  Beitrage,  1;  Mayer,  Arch.  f.  (Anat.  u.) 
Physiol.,  1902,  Zeitschr.  f.  klin.  Med.,  47,  and  Zeitschr.  f.  physiol.  Chem.,  29,  32;  Scholz, 
ibid.,  38;    Ellinger,  ibid.,  39. 

2  Harnack,  ibid.,  29;  Scholz,  I.  c. ;  Moraczewski,  Centralbl.  f.  innere  Med.,  1903. 

3  See  JafT6,  Pfluger's  Arch.,  3;  Senator,  Centralbl.  f.  d.  med.  Wissensch.,  1877; 
G.  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  12  (contains  older  literature);  also 
Berl.  klin.  Wochenschr.,  1892. 

*  Baumann  with  Brieger,  Zeitschr.  f.  physiol.  Chem.,  3;  with  Thesen,  ibid.,  23. 
5JafT6,  Pfluger's  Arch.,  3;    Obermayer,  Wien.  klin.  Wochenschr.,  1890;    Porcher 
and  Hervieux,  Zeitschr.  f.  physiol.  Chem.,  39. 


SKA  TOXYLSULPHURIC  ACID. 

The  acid  urine  (if  alkaline  it  must  be  acidified  with  acetic  acid) 
(Ellixgkk)  is  precipitated  with  basic  lead  acetate,  1  c.  c.  for  every  10  c.  c. 
of  the  urine.  20  c.  c.  of  the  filtrate  are  treated  in  a  test-tube  with  an  equal 
volume  of  pure  concentrated  hydrochloric  acid  (specific  gravity  1.19) 
which  contains  2  A  grama  ferric  chloride  in  the  liter  and  2-3  c.  c.  chloro- 
form added  and  immediately  thoroughly  shaken.  The  chloroform  is 
colored  hereby  more  or  less  blue,  depending  upon  the  amount  of  indican. 
besides  indigo  blue  we  may  also  have  indigo  red  produced,  whose  formation 
has  been  explained  in  various  ways.  The  quantity  of  indigo  red  becomes 
greater  the  slower  the  oxidation  takes  place,  and  especially  when  the 
decomposition  takes  place  in  the  warmth  (see  the  works  of  Rosin,  Bouma, 
Wang,  .Maillard,  and  Ellinger1). 

The  chloroform  solution  of  indigo  obtained  in  the  indican  test  may  be 
used  in  the  quantitative  colorimetric  determination  by  comparison  with  a 
solution  of  indigo  in  chloroform  of  known  strength  (Krauss  and  Adrian3). 
Wang  and  others  convert  the  indigo  into  indigo-sulphonic  acid  by  con- 
centrated sulphuric  acid  and  titrate  with  potassium  permanganate.  It  is 
still  undecided  as  to  the  surest  and  most  trustworthy  method  for  the  deter- 
mination of  indican  and  especially  as  to  the  question  how  the  indigo  resi- 
due is  to  be  washed  (see  Wang,  Bouma,  and  Ellinger)  and  for  this  reason 
we  will  only  refer  to  the  works  cited  above. 

Indol  seems  also  to  pass  into  the  urine  as  a  glucuronic  acid,  indoxyl- 
glucuronic  acid  (Schmiedeberg).  Such  an  acid  has  been  found  in  the  urine 
of  animals  after  the  administration  of  the  sodium-salt  of  o-nitro-phenyl- 
propiolic  acid  (G.  Hoppe-Seyler  3). 

CH 

/\ 

HC      C-C.CH, 
Skatoxyl-sulphuric  Acid,   C9H9NS04=  ,   has  not 

HC       C    C.O.S02OH 

CH  NH 
been  positively  prepared  as  a  constituent  of  normal  urine,  while  Otto  has 
once  prepared  its  alkali  salt  from  diabetic  urine.  Perhaps  skatoxyl  occurs 
in  normal  urine  as  a  conjugated  glucuronate  (Mayer  and  Neuberg  *) ,  and 
it  is  believed  that  the  urine  contains  a  skatol-chromogen  from  which  red  and 
reddish-violet  coloring-matters  are  obtained  by  decomposition  with  strong 
acids  and  an  oxidizing  agent. 

Skatoxyl-sulphuric  acid  originates,  if  it  exists  in  the  urine,  from  skatol 


1  Rosin,  Virchow's  Arch.,  123;  Bouma,  Zeitschr.  f.  physiol.  Chem.,  27,  30,  32, 
39;  Wang,  ibid.,  25,  27,  28;  Ellinger,  ibid.,  38;  Maillard,  Bull.  soc.  chim.,  Paris  (3), 
29,  and  Compt.  rend.,  136. 

'  Krauss,  Zeitschr.  f.  physiol.  Chem.,  18;  Adrian,  ibid.,  19;   Wang,  ibid.,  25. 

•Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  14;  G.  Hoppe-Seyler,  Zeitschr.  f. 
physiol.  Chem.,  7  and  8. 

4  Otto,  Pfluger's  Arch.,  33;  Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29. 


510  URINE. 

which  is  formed  by  putrefaction  in  the  intestine,  and  which  is  then 
coupled  with  sulphuric  acid  after  oxidation  into  skatoxyl.  That  skatol 
introduced  into  the  body  passes  partly  as  an  ethereal-sulphuric  acid  into 
the  urine  has  been  shown  by  Brieger.  Indol  and  skatol  act  differently,  at 
least  in  dogs ;  indol  producing  a  considerable  amount  of  ethereal-sulphuric 
acid,  while  skatol  gives  only  a  small  quantity  (Mester  l). 

The  potassium-salt  of  skatoxyl-sulphuric  acid  is  crystalline;  it  dissolves 
in  water,  but  with  difficulty  in  alcohol.  A  watery  solution  becomes  deep 
violet  with  ferric  chloride  and  red  with  concentrated  nitric  acid.  The  salt 
is  decomposed  by  concentrated  hydrochloric  acid  with  the  separation  of  a 
red  precipitate.  The  nature  of  this  red  coloring-matter  produced  by  the 
decomposition  of  skatoxyl-sulphuric  acid  is  not  well  known;  neither  has  the 
relationship  existing  between  this  and  other  red  coloring-matters  in  the 
urine  been  decided.  On  distillation  with  zinc-dust  the  skatol-chromogen 
yields  skatol. 

Urines  containing  skatoxyl  are  colored  dark  red  to  violet  by  Jaffe's 
indican  test  even  on  the  addition  of  hydrochloric  acid;  with  nitric  acid  they 
are  colored  cherry-red,  and  red  on  warming  with  ferric  chloride  and  hydro- 
chloric acid.  The  coloring-matter  which  yields  skatol  with  zinc-dust  may 
be  removed  from  the  urine  by  ether.  Urines  rich  in  skatoxyl  darken  from 
the  surface  downward  when  allowed  to  stand  in  the  air,  and  may  become 
reddish,  violet,  or  nearly  black.  Rosin  2  is  of  the  opinion  that  no  skatol- 
chromogen  exists  in  human  urine,  and  that  the  observations  made  hereto- 
fore were  due  to  a  confusion  with  indigo  red  or  urorosein.  We  find  also 
sometimes  statements  as  to  a  red  "skatol  pigment,"  which  like  urorosein 
is  soluble  in  amyl  alcohol,  but  insoluble  in  chloroform,  and  whose  nature 
is  questionable.  Only  the  formation  of  skatol  by  distillation  with  zinc 
powder  can  be  considered  as  a  positive  proof  as  to  the  skatol  nature  of  a 
pigment. 

Salkowski3  has  demonstrated  that  the  occurrence  of  skatol-carbonic  acid, 
C,,H8.N.COOH,  in  normal  urine  is  probable.  Thi  is  also  a  product  of  putrefaction. 
When  introduced  into  the  animal  body  this  acid  reappears  unchanged  in  the 
urine.  With  hydrochloric  acid  and  very  dilute  ferric-chloride  solution  it  gives  an 
intense  violet  color  to  the  solution.  The  reaction  responds  with  a  watery  solu- 
tion containing  1  :  10,000  of  skatol-carbonic  acid. 

Aromatic  Oxyacids.  In  the  putrefaction  of  proteids  in  the  intestine, 
paraoxyphenyl-acetic  acid,  C6H4(OH).CH2COOH,  and  paraoxyphenyl-pro- 
pionic  acid,  CaH4(OH).C2H4.COOH,  are  formed  from  tyrosin  as  an  interme- 


1  Brieger,   Ber.  d.  deutsch.   chem.  Gesellsch.,  12,  and  Zeitschr.  f.  physiol.  Chem., 
4,  414;  Mester,  ibid.,  12. 

2  Virchow's  Arch.,  123. 

3  Zeitschr.  f.  physiol.  Chem.,  9. 


AROMATIC  OX  Y  AC  IDS.  511 

diate  step,  and  these  in  great  part  pass  unchanged  into  the  urine.    The 

quantity  of  these  acids  is  usually  very  small.     They  are  increased  under  the 

same  conditions  as  the  phenols,  especially  in  acute  phosphorus  poisoning, 

in  which  the  increase  is  considerable.     A  small  portion  of  these  oxyacids 

is  combined  with  sulphuric  acid. 

Besides  these  two  oxyacids  which  regularly  occur  in  human  urine  we 

sometimes  have  other  oxyacids  in  urines.     To  these  belong  homogentisic 

acid  and  uroleucic  acid,  which  form  the  specific  constituents  of  the  urine 

in  most  cases  of  alcaptonuria,  oxymandelic  acid,  found  by  Schultzen  and 

Riess  in  urine  in  acute  atrophy  of  the  liver,  oxyhydroparacoumaric  acid, 

found  by  Blendermann  in  the  urine  on  feeding  rabbits  with  tyrosin,  gallic 

acid,  which,  according  to  Baumann,1  sometimes  appears  in  horse's  urine, 

and  kynurenic  acid  (oxyquinolincarbonic  acid),  which  up  to  the  present 

time  has  been  found  only  in  dog's  urine.     Although  all  these  acids  do  not 

belong  to  the  physiological  constituents  of  the  urine,  still  they  will  be 

treated  in  connection  with  these. 

OTT 
Paraoxy phenylacetic  Acid,  C8H803  =  C„H4 < ^ ^  COOH'  anc* 

p-Oxyphenylpropionic     Acid      (Hydroparacoumaric     Acid),      C„H10O3= 

OH 
C„H4  <  prf  pxr  poqtj  f  are  crystalline  and  are  both  soluble  in  water  and 

in  ether.    The  first  melts  at  148°  C.  and  the  other  at  125°  C.    Both  give 
a  beautiful  red  coloration  on  being  warmed  with  Millon  's  reagent. 

To  detect  the  presence  of  these  oxyacids  proceed  in  the  following  way  (Bau- 
mann) :  Warm  the  urine  for  a  while  on  the  water-bath  with  hydrochloric  acid 
in  order  to  drive  off  the  volatile  phenols.  After  cooling  shake  three  times  with 
ether,  and  then  shake  the  ethereal  extracts  with  dilute  soda  solution,  which  dis- 
solves the  oxyacids,  while  the  residue  of  the  phenols  which  ar  soluble  in  ether 
remains.  The  alkaline  solution  of  the  oxyacids  is  now  faintly  acidified  with  sul- 
phuric acid,  shaken  again  with  ether,  the  ether  removed  and  allowed  to  evaporate, 
the  residue  dissolved  in  a  little  water,  and  the  solution  tested  with  Millon's 
reagent.  The  two  oxyacids  are  best  differentiated  by  their  different  melting- 
points.  The  reader  is  referred  to  other  works  for  the  method  of  isolating  and 
separating  these  two  oxyacids. 

Homogentisic  Acid  (Dioxyphenylacetic  Acid),  C8H804  = 

/OH(1) 
C„H3^— OH(4)  .    This  acid  was  detected  by  Wolkow  and  Baumann. 

\CH2.COOH(5> 
They  isolated  it  from  the  urine  in  a  case  of  alcaptonuria  (see  below)  and 
showed    that    the    characteristics   of    so-called    alcaptonuric  urine  in  this 
case  were   due   to   this   acid.     This  acid  has  later  been   found   in  other 

'Schultzen  and  Riess,  Chem.  Centralbl.,  1869;  Blendermann,  Zeitschr.  f.  physiol. 
Chem.,  6,  267;  Baumann,  ibid.,  6,  193. 


512  URINE. 

cases  of  alcaptonuria  by  Embden,  Garnier  and  Voirin,  Ogden,  and 
others.  Glycosuric  acid,  isolated  from  alcaptonuric  urine  first  by  Marshall 
and  then  by  Geyger,1  seems  to  be  in  part  identical  with  homogentisic  acid. 

The  quantity  of  acid  eliminated  is  increased  by  food  rich  in  proteid.  On 
the  ingestion  of  tyrosin  by  persons  with  alcaptonuria,  Wolkow  and 
Baumantn  and  Embden  observed  a  greater  quantity  of  homogentisic 
acid,  and  Falta  and  Langstein  2  found  the  same  after  phenylalanin. 
These  last-mentioned  investigators  found  that  the  calculated  quantity  of 
tyrosin  from  the  various  proteids  was  not  sufficient  to  account  for  the 
homogentisic  acid,  and  hence  they  consider  the  phenylalanin  as  a  source 
for  this  acid.  Of  the  1-phenylalanin  about  90  per  cent,  and  of  the  racemic 
phenylalanin  about  50  per  cent  was  eliminated  as  homogentisic  acid. 
Wolkow  and  Baumann  explain  the  formation  of  homogentisic  acid  from 
tyrosin  by  an  abnormal  fermentation  in  the  intestine.  According  to 
Langstein  and  Falta  alcaptonuria  seems  to  be  an  anomaly  of  the  inter- 
mediary metabolism. 

Garrod,3  who  has  observed  several  cases  of  alcaptonuria,  has  also  tab- 
ulated about  forty  cases  of  alcaptonuria  which  he  finds  in  the  literature. 
From  this  he  shows  that  the  anomaly  of  the  proteid  metabolism  occurs 
often  in  males  and  in  females,  and  also  that  blood  relationship  of  the 
parents  (first  cousins)  predisposes  alcaptonuria. 

On  fusing  homogentisic  acid  with  alkali  it  yields  gentisic  acid  (hydro- 
quinone-carbonic  acid)  and  hydroquinone.  When  introduced  into  the  intes- 
tine of  the  dog  a  part  is  converted  into  toluhydroquinone,  which  is  elimi- 
nated in  the  form  of  an  ethereal  sulphuric  acid.  Homogentisic  acid  has 
also  been  prepared  synthetically  by  Baumann  and  Frankel,4  starting  with 
gentisic  aldehyde. 

Homogentisic  acid  crystallizes  with  1  mol.  of  water  in  large,  trans- 
parent prismatic  crystals,  which  become  non-transparent  at  the  tempera- 
ture of  the  room  with  the  loss  of  water  of  crystallization.  They  melt  at 
146.5-147°  C.  They  are  soluble  in  water,  alcohol,  and  ether,  but  nearly 
insoluble  in  chloroform  and  benzene.  Homogentisic  acid  is  optically  in- 
active and  non-fermentable.  Its  watery  solution  has  the  properties  of  so- 
called  alcaptonuric  urine.  It  becomes  greenish  brown  from  the  surface 
downward  on  the  addition  of  very  little  caustic  soda  or  ammonia  with 
excess  of  oxygen,  and  on  stirring  it  quickly  becomes  dark  brown  or  black. 

'Wolkow  and  Baumann,  Zeitschr.  f.  physiol.  Chem.,  15;  Embden,  ibid.,  17  and 
18;  Garnier  and  Voirin,  Arch,  de  Physiol.  (5),  4;  Ogden,  Zeitschr.  f.  physiol.  Chem., 
20;  Marshall,  Maly's  Jahresber.,  17;  Geyger,  cited  from  Embden,  1.  c,  18. 

1  Zeitschr.  f.  physiol.  Chem.,  37. 

3  Med.  chirurg.  Transact.,  1899  (where  all  known  cases  are  tabulated);  also  The 
Lancet,  1901  and  1902. 

*  Zeitschr.  f.  physiol.  Chem.,  20. 


AROMATIC  OX Y ACIDS.  513 

It  reduces  alkaline  copper  solutions  with  even  slight  heat,  and  ammoniacal 
silver  solutions  immediately  in  the  cold.  It  does  not  reduce  alkaline  bis- 
inut  h  s<  .lilt  ions.  It  gives  a  lemon-colored  precipitate  with  MiLLON  's  reagent, 
which  becomes  light  brick-red  on  warming.  Ferric  chloride  gives  to  the 
solution  a  blue  color  which  soon  disappears.  On  boiling  with  concentrated 
ferric  chloride  solution  an  odor  of  quinone  develops.  With  benzoyl  chloride 
and  caustic  soda  in  the  presence  of  ammonia  we  obtain  the  amide  of  diben- 
zoylhomogentisic  acid,  which  melts  at  204°  C,  and  which  can  be  used  in  the 
Isolation  of  the  acid  from  the  urine  and  also  for  its  detection  (Orton  and 
Garrod).  Among  the  salts  of  this  acid  must  be  mentioned  the  lead  salt 
containing  water  of  crystallization  and  31.79  per  cent  Pb.  This  salt  melts 
at  214-215°  C. 

In  order  to  prepare  the  acid,  heat  the  urine  to  boiling,  add  5  grams  of  lead 
acetate  for  every  100  c.  c,  filter  as  soon  as  the  lead  acetate  has  dissolved, 
and  allow  the  filtrate  to  stand  in  a  cool  place  for  twenty-four  houis  until  it 
crystallizes  (Garrod).  The  dried,  powdered  lead  salt  is  suspended  in  ether 
and  decomposed  by  H2S.  After  the  spontaneous  evaporation  of  the  ether 
the  acid  is  obtained  in  nearly  colorless  crystals  (Orton  and  Garrod  *). 

In  regard  to  the  quantitative  estimation  we  proceed  according  to  the  sug- 
gestion of  Baumann  by  titrating  the  acid  with  a  N/10  silver  solution.  As  regards 
details  of  this  method  the  reader  is  referred  to  the  works  of  Bai.mann,  C.  Th. 
Morner  and  Mittlebach.     Deniges  2  has  sugges  ed  another  method. 

Uroleucic  acid,  C,jH10O5,  is,  according  to  Huppert,  probably  a  dioxvphenyl- 
lactic  acid,  C9H3(OH  2.Cil2.CH(OH).COOH.  This  acid  was  first  prepared  by  Kirk  3 
from  the  urine  of  children  with  alcap  onuria,  which  also  contained  homogentisic 
acid.  It  melts  at  130-133°  C.  Otherwise,  in  regard  to  its  behavior  with  alkalies, 
with  access  of  air,  and  also  with  alkaline  copper  solution;  and  ammoniacal  silver 
solutions,  and  also  Millon's  reagent,  it  is  similar  to  homogentisic  acid. 

Oxymadelic  acid,  paraoxyphenylglycolic  acid,  C8H804,  HO.C6H4.CH(OH)COOH, 
is,  as  above  stated,  found  in  the  urine  in  acute  atrophy  of  the  liver.  The  acid 
crystallizes  in  silky  needles.  It  melts  at  162°  C,  dissolves  readily  in  hot  water, 
less  in  cold  water,  and  readily  in  alcohol  and  ether,  but  not  in  hot  b;nzene. 
It  is  precipitated  by  basic  lead  acetate,  but  not  by  lead  acetate. 

CH    COH 

Kynurenic  acid  (^-oxy-3-quinolincarbonic  acid) ,  C10H7XO,  -  HC     C      C.COOH, 

I     I!     I 

HC     C     CH 


CH   N 

has  only  been  found  thus  far  in  dog's  urine;  its  quantity  is  increased  by 
meat  feeding.  According  to  the  observations  of  Glaessner  and  Langstf.iv/ 
the  mother-substance  seems  to  be  contained  among  the  products  of  pancreatic 

1  Orton  and  Garrod,  Journ.  of  Physiol.,  27;   Garrod,  ibid.,  23. 
:  Mittlebach,  Deutsch.  Arch.  f.  klin.  Med.,  71  (which  contains  the  work  of  Baumann 
and  Morner);   Deniges,  Chem.  Centralbl.,  1897,  1,  338. 

3  Huppert,  Zeitschr.  f.  physiol.  Chem.,  23;  Kirk,  Brit.  med.  Journ.,  1886  and  1888, 
Arch.  f.  Anat.  u.  Physiol.,  23. 

4  Hofmeister's  Beitrage,  1. 


514  URINE. 

digestion  which  are  soluble  in  alcohol  and  precipitable  by  acetone.  The  acid 
is  crystalline,  dissolves  in  cold  water,  rather  well  in  hot  alcohol,  and  yields  a 
barium  salt  which  crystallizes  in  triangular,  colorless  plates.  On  heating  it 
melts  and  decomposes  into  C02  and  kynurin.  On  evaporation  to  dryness  on 
the  water-bath  with  hydrochloric  acid  and  potassium  chlorate  a  reddish  residue 
is  obtained  which  becomes  first  brownish  green  and  then  emerald-green  on  adding 
ammonia  (Jaffe's  reaction1). 

Urinary  Pigments  and  Chromogens.  The  yellow  color  of  normal  urine 
depends  perhaps  upon  several  pigments,  but  in  greatest  part  upon  urochrome. 
Besides  this  the  urine  seems  to  contain  a  very  small  quantity  of  hcemato- 
porphyrin  as  a  regular  constituent.  Uroerythrin  also  is  of  frequent 
occurrence  in  normal  urine,  but  not  always.  Finally,  the  excreted  urine 
when  exposed  to  the  action  of  light  regularly  contains  a  yellow  pigment, 
urobilin,  which  is  derived  from  a  chromogen,  urobilinogen,  by  the  action 
of  light  (Saillet)  and  air  (Jaffe,  Disque,2  and  others).  Besides  this 
chromogen,  urine  contains  various  other  bodies  from  which  coloring-matters 
may  be  produced  by  the  action  of  chemical  agents.  Humin  substances 
(perhaps  in  part  from  the  carbohydrates  of  the  urine)  may  be  formed  by 
the  action  of  acids  (v.  Udranszky)  without  regard  to  the  fact  that  such  sub- 
stances may  sometimes  originate  from  the  reagents  used,  as  from  impure 
amyl  alcohol  (v.  Udranszky  3).  To  these  humin  bodies  developed  by  the 
action  of  acid  in  normal  urine  when  exposed  to  the  air,  must  be  added 
the  urophain  of  Heller,  the  various  uromelanins  and  other  bodies  de- 
scribed by  different  investigators  (Pl6sz,  Thudichum,  Schunk4).  In- 
digo-blue (uroglaucin  of  Heller,  urocyanin,  cyanurin,  and  other 
coloring-matters  of  older  investigators  5)  is  split  off  from  the  indoxyl- 
sulphuric  acid  or  indoxyl-glucuronic  acid.  Red  coloring-matters  may  be 
formed  from  the  conjugated  indoxyl  and  skatoxyl  acids,  and  urohodin 
(Heller),  urorubin  (Pl6sz),  urohcematin  (Harley),  and  perhaps  also 
urorosein  (Nencki  and  Sieber  6)  probably  have  such  an  origin. 

We  cannot  discuss  more  in  detail  the  different  coloring-matters  obtained 
as  decomposition  products  from  normal  urine.  Haematoporphyrin  has 
already  been  referred  to  in  a  previous  chapter  (VI)  and  will  best  be  de- 
scribed in  connection  with  the  pathological  pigments.  It  only  remains  to 
describe  urochrome,  urobilin,  and  uroerythrin. 

1  Zeitschr.  f.  physiol.  Chem.,  7.  In  regard  to  kynurenic  acid,  see  also  Huppert- 
Neubauer,  10.  Aufl.,  and  Mendel  and  Jackson,  Amer.  Journ.  of  Physiol.,  2;  Mendel 
and  Schneider,  ibid.,  5;  Camps,  Zeitschr.  f.  physiol.  Chem.,  33. 

2  Jaffe\  Centralbl.  f.  d.  med.  Wissensch.  1868  and  1869,  and  Virchow's  Arch.,  47; 
Disque\  Zeitschr.  f.  physiol.  Chem.,  2;  Saillet,  Revue  de  medecine,  17,  1897. 

3  v.  Udrdnszky,  Zeitschr.  f.  physiol.  Chem.,  11,  12,  and  13. 

*  Plosz,  Zeitschr.  f.  physiol.  Chem.,  8;  Thudichum,  Brit.  med.  Journ.,  201,  and 
Journ.  f.  prakt.  Chem.,  104;   Schunk,  cited  from  Huppert-Neubauer,  10.  Aufl.,  509. 

5  See  Huppert-Neubauer,  161. 

8  In  regard  to  this  and  other  red  pigments,  see  Huppert-Neubauer,  593  and  597; 
Nencki  and  Sieber,  Journ.  f.  prakt.  Chem.  (2),  26. 


UROCHROME.     UROBILIN.  515 

Urochrome  is  the  name  given  by  Garrod  to  the  yellow  pigment  of  the 
urine.  Thudichum  l  had  previously  given  the  same  name  to  a  less  pure 
pigment  isolated  by  himself.  According  to  Garrod  urochrome  is  free  from 
iron,  but  contains  nitrogen.  It  stands,  it  seems,  in  close  relationship  to 
urobilin,  as  Garrod  has  obtained  a  urobilin-like  pigment  by  the  action  of 
aldehyde  on  urochrome,  and  Riva  2  claims  that  urobilin  yields  a  body  similar 
to  urochrome  on  careful  oxidation  with  permanganate.  According  to  Gar- 
rod urobilin  can  be  converted  into  urochrome  by  evaporating  its  aqueous 
solution  containing  some  ether  on  the  water-bath.  The  fact  that  uro- 
chrome can  be  transformed  into  urobilin  by  means  of  active  acetaldehyde 
may  be  used,  according  to  Garrod,  as  a  means  of  detecting  urochrome. 

Urochrome  is,  according  to  Garrod,  amorphous,  brown,  very  readily 
soluble  in  water  and  ordinary  alcohol,  but  less  soluble  in  absolute  alcohol. 
It  dissolves  but  slightly  in  acetic  ether,  amyl  alcohol,  and  acetone,  while  it 
is  insoluble  in  ether,  chloroform,  and  benzene.  Urochrome  is  precipitated 
by  lead  acetate,  silver  nitrate,  mercuric  acetate,  phosphotungstic  and  phos- 
phomolybdic  acids.  On  saturating  the  urine  with  ammonium  sulphate  a 
great  part  of  the  urochrome  remains  in  solution.  It  does  not  show  any 
absorption-bands  and  does  not  fluoresce  after  the  additionjof  ammonia  and 
zinc  chloride.  Urochrome  is  very  readily  decomposed,  with  the  formation 
of  brown  substances,  by  the  action  of  acids.  According  to  Klemperer,3 
urochrome  contains  4.2  per  cent  nitrogen. 

Urochrome  can  be  prepared  according  to  a  rather  complicated  method 
which  is  based  upon  the  fact  that  the  substance  remains  in  great  part  in 
solution  on  saturating  the  urine  with  ammonium  sulphate.  If  the  proper 
quantity  of  alcohol  is  added  to  the  filtrate,  a  clear,  yellow  alcoholic  layer 
forms  on  the  salt  solution,  which  contains  the  urochrome  and  which  can 
be  used  for  the  further  preparation  of  the  urochrome  (see  Garrod,  1.  c). 
Klkmperer,  on  the  contrary,  removes  the  pigment  from  the  urine  by 
means  of  animal  charcoal,  washes  it  with  water  to  remove  the  indican 
and  other  bodies,  and  then  extracts  with  alcohol  and  uses  this  alcoholic 
extract  for  the  further  purification  according  to  Garrod. 

The  urochrome  can  be  quantitatively  estimated,  according  to  Klem- 
perer, by  a  colorimetric  method,  using  a  solution  of  true  yellow  G.  If 
0.1  gram  of  this  dye  is  dissolved  in  1  liter  of  water  and  5  c.  c.  of  this  solu- 
tion diluted  to  50  c.  c.  with  water,  then  this  solution  has  the  same  color  and 
shade  as  a  0.1  per  cent  urochrome  solution.  The  urine  must  be  diluted 
with  water  until  it  has  the  same  depth  of  color.  The  comparison  is  per- 
formed in  vessels  with  parallel  walls. 

Urobilin  is  the  pigment  first  isolated  from  the  urine  by  Jaffe,4  and 
which  is  characterized  by  its  strong  fluorescence  and  by  its  absorption- 

1  Garrod,  Proceed.  Roy.  Soc.,  55;   Thudichum,  1.  c. 

'Garrod,  Journ.  of  Physiol.,  21  and  29;   Riva,  cited  from  Huppert-Xeubauer   524. 

3  Berlin,  klin.  Wochenschr.,  40. 

4  Centralbl.  f  d.  med.  Wissensch.,  1SCS  and  1869,  and  Virchow's  Arch.,  4" 


516  URINE. 

spectrum.  Various  investigators  have  prepared  from  the  urine  by  different 
methods  pigments  which  differed  slightly  from  each  other  but  behaved 
essentially  like  Jaffe's  urobilin.  Thus  different  urobilins  have  been 
suggested,  such  as  normal,  febrile,  physiological,  and  pathological  urobilins.1 
The  possibility  of  the  occurrence  of  different  urobilins  in  the  urine  cannot 
be  denied;  but  as  urobilin  is  a  readily  changeable  body  and  difficult  to 
purify  from  other  urinary  pigments,  the  question  as  to  the  occurrence  of 
different  urobilins  must  still  be  considered  open.  According  to  Saillet  2 
no  urobilin  exists  originally  in  human  urine,  but  only  the  mother-substance 
of  the  same,  urobilinogen,  from  which  the  urobilin  is  formed  in  the  excreted 
urine  by  the  influence  of  light. 

Urobilin-like  bodies,  so-called  urobilinoids,  have  been  prepared  from 
bile-pigments  as  well  as  blood-pigments,  and  indeed  by  oxidation  as  well  as 
reduction.  Maly  obtained  his  hydrobilirubin  by  the  reduction  of  bilirubin 
with  sodium  amalgam,  and  Disque  obtained  a  product  which  is  still  more 
similar  to  urobilin,  while  Stokvis  prepared  by  the  oxidation  of  cholecyanin 
with  a  little  lead  peroxide  a  choletelin  which  acted  very  much  like  urobilin. 
Hoppe-Seyler,  Le  Nobel,  Nencki  and  Sieber  have  obtained  urobilinoid 
bodies  by  the  roluction  of  hsematin  and  hsematoporphyrin  with  tin  or  zinc 
and  hydrochloric  acid,  while  MacMunn  3  obtained  by  the  oxidation  of 
hsematin  with  hydrogen  peroxide  in  alcohol  containing  sulphuric  acid  a 
pigment  which  seemed  to  be  identical  with  urinary  urobilin.  It  is  apparent 
that  all  these  urobilins  cannot  be  identical. 

Many  investigators  declare  that  urobilin  is  identical  with  hydrobilirubin, 
but  according  to  the  researches  of  Hopkins  and  Garrod  4  this  view  is  not 
correct,  because,  irrespective  of  other  small  differences,  each  body  has 
an  essentially  distinct  composition.  Hydrobilirubin  contains  C  64.68, 
H  6.93,  N  9.22  (Maly),  while  urinary  urobilin,  on  the  contrary,  contains 
C  63.46,  H  7.67,  N  4.09  per  cent.  The  urobilin  from  fseces,  stercobilin, 
has  the  same  composition  as  urinary  urobilin  with  4.17  per  cent  nitrogen. 

Urinary  urobilin  may  not  be  identical  with  hydrobilirubin,  but  this  does 
not  eliminate  the  possibility  that  urobilin,  according  to  the  generally 
admitted  view,  is  derived  from  bilirubin  (although  not  by  simple  reduction 
and  taking  up  water)  in  the  intestine.     Several  physiological  as  well  as 

1  See  MacMunn,  Proc.  Roy.  Soc,  31  and  35;  Ber.  d.  deutsch.  chem.  Gesellsch..  14, 
and  Journ.  of  Physiol.,  6  and  10;  Bogomoloff,  Maly's  Jahresber,  22;  Eichholz,  Journ. 
of  Physiol.,  14;   Ad.  Jolles,  Pfluger's  Arch.,  61. 

2  Revue  de  medecine,  19,  1897. 

3  Maly,  Ann.  d.  Chem.  u.  Pharm.,  163;  Disqu6,  Zeitschr.  f.  physiol.  Chem.,  2; 
Stokvis,  Centralbl.  f.  d.  med.  Wissensch.,  1873,  211  and  449;  Hoppe-Seyler,  Ber  d. 
deutsch.  chem.  Gesellsch.,  7;  Le  Nobel,  Pfliiger's  Arch.,  40;  Nencki  and  Sieber, 
Monatshefte  f.  Chem.,  9;  and  Arch.  f.  exp.  Path.  u.  Pharm.,  24;  MacMunn,  Proc  Roy. 
Soc,  31. 

4  Journ.  of  Physiol.,  22. 


UROBILIN.  517 

clinical  observations  '  speak  fur  this  view,  among  which  we  must  mention 
the  regular  appearance  in  the  intestinal  tract  (if  stercobilin,  undoubtedly 
derived  from  the  bile-pigments  and  having  the  same  composition  as  urinary 
urobilin,  the  absence  of  urobilin  in  the  urine  of  new-born  infants  and  also 
on  the  complete  removal  of  bile  from  the  intestine,  as  well  as  the  increased 
elimination  of  urobilin  with  strong  intestinal  putrefaction.  On  the  other 
hand,  there  are  investigators  who,  basing  their  opinion  on  clinical  observa- 
tions, deny  the  intestinal  origin  of  urobilin  and  claim  that  the  urobilin  is 
derived  from  a  transformation  of  the  bilirubin  elsewhere  than  in  the  intes- 
tine, by  an  oxidation  of  the  bile-pigment  or  by  a  transformation  of  the 
blood-pigments.1  The  possibility  of  a  different  mode  of  formation  of  uri- 
nary urobilin  in  disease  is  not  to  be  denied;  but  there  is  no  doubt  that 
this  pigment  is  formed  from  the  bile-pigments  in  the  intestine  under  physio- 
logical  conditions. 

The  quantity  of  urobilin  in  the  urine  under  physiological  conditions  is 
very  variable.  Saillet  found  30-130  milligrams  and  G.  Hoppe-Seyler. 
80-140  milligrams  in  one  day's  urine. 

There  are  numerous  observations  on  the  elimination  of  urobilin  in 
disease,  especially  by  Jaffe,  Disque,  Dreyfuss-Brissac,  Gerhardt,  G. 
Hoppe-Seyler,'  and  others.  The  quantity  is  increased  in  hemorrhage  and 
in  such  diseases  where  the  blood-corpuscles  are  destroyed,  as  is  the  case 
after  the  action  of  certain  blood-poisons,  such  as  antifibrine  and  antipyrine. 
It  is  also  increased  in  fevers,  heart- troubles,  lead  colic,  atrophic  cirrhosis 
of  the  liver,  and  is  especially  abundant  in  so-called  urobilin  icterus. 

The  properties  of  urobilin  may  be  different,  depending  upon  the  method 
of  preparation  and  the  character  of  the  urine  used;  therefore  only  the 
most  important  properties  will  be  given.  Urobilin  is  amorphous,  brown, 
reddish-brown,  red,  or  reddish-yellow,  depending  upon  method  of  prepara- 
tion. It  dissolves  readily  in  alcohol,  amyl  alcohol,  and  chloroform,  but 
readily  in  ether  or  acetic  ether.  It  is  less  soluble  in  water,  but  the 
solubility  is  augmented  by  the  presence  of  neutral  salts.  It  may  be  com- 
pletely precipitated  from  the  urine  by  saturating  with  ammonium  sulphate, 
especially  after  the  addition  of  sulphuric  acid  (Mehy  *).  It  is  soluble  in 
alkalies,  and  is  precipitated  from  the  alkaline  solution  by  the  addition  of 

xSee  Fr.  Miiller,  Schles.  Gesellsch.  f.  vaterl.  Kultur,  1892;  D.  Gerhardt,  "Ueber 
lly.lrobilirubin  und  seine  Bezieh.  zum  Ikterus"  (Inaug.-Diss.,  Berlin,  1SS9);  Beck, 
Wien.  klin.  AYochenschr.,  1S95;   Harley,  Brit.  Med.  Journ.,  1896. 

2  In  regard  to  the  various  theories  as  to  the  formation  of  urobilin,  see  Harley, 
Brit.  Med.  Journ.,  1896;  A.  Katz,  "\Yien.  med.  Wochenschr. ,  1891,  Xos.  2S-32;  Grimm, 
Virchow'a  Arch.,  132;   Zoja,  Conferenze  cliniche  italiane,  Ser.  la,  1. 

3  In  regard  to  the  literature  on  this  subject  we  refer  the  reader  to  D.  Gerhardt, 
"Ueber  Hydrobilirubin  und  seine  Beziehungen  zum  Ikterus"  (Berlin,  1889),  and 
also  G.  Hoppe-Seyler,  Yirchow's  Arch.,  124. 

4  Journ.  de  Pharru.  et  Chini.,  1S78,  cited  from  Maly's  Jahresber.,  8. 


518  URINE. 

acid.  It  is  partly  dissolved  by  chloroform  from  an  acid  (watery-alcoholic) 
solution;  alkali  solutions  remove  the  urobilin  from  the  chloroform.  The 
neutral  or  faintly  alkaline  solutions  are  precipitated  by  certain  metallic 
salts  (zinc  and  lead),  but  not  by  others,  such  as  mercuric  sulphate.  Uro- 
bilin is  precipitated  from  the  urine  by  phosphotungstic  acid.  It  does  not 
give  Gmelin's  test  for  bile-pigments.  It  gives,  on  the  contrary,  a  reaction 
which  may  be  mistaken  for  the  biuret  test,  by  the  action  of  copper  sulphate 
and  alkali.1 

Neutral  alcoholic  urobilin  solutions  are  in  strong  concentration  brownish- 
yellow,  in  great  dilution  yellow  or  rose-colored.  They  have  a  strong  green 
fluorescence.  The  acid  alcoholic  solutions  are  brown,  reddish-yellow,  or  rose- 
red,  according  to  concentration.  They  are  not  fluorescent,  but  show  a 
faint  absorption-band,  y, between  b  and  F,  which  borders  onF,  or  in  greater 
concentration  extends  over  F.  The  alkaline  solutions  are  brownish-yellow, 
yellow,  or  (the  ammoniacal)  yellowish-green,  according  to  concentration. 
If  some  zinc-chloride  solution  is  added  to  an  ammoniacal  solution  of  the  pig- 
ment it  becomes  red  and  shows  a  beautiful  green  fluorescence.  This  solution, 
as  also  that  made  alkaline  with  fixed  alkalies,  shows  a  darker  and  more 
sharply  defined  band,  d,  between  b  and  F,  almost  midway  between  E  and  F. 
If  a  sufficiently  concentrated  solution  of  urobilin  alkali  is  carefully  acidi- 
fied with  sulphuric  acid  it  becomes  cloudy  and  shows  a  second  band  exactly 
at  E  and  connected  with  y  by  a  shadow  (Garrod  and  Hopkins,  Saillet  2). 

Urobilinogen  is  colorless  or  is  only  slightly  colored.  Like  urobilin,  it  is 
precipitated  from  the  urine  by  saturating  with  ammonium  sulphate.  Ac- 
cording to  Saillet  it  may  be  extracted  by  acetic  ether  from  urine  acidi- 
fied with  acetic  acid.  It  dissolves  also  in  chloroform,  ethyl  ether,  and 
amyl-alcohol.  It  shows  no  absorption-bands,  and  is  readily  converted  into 
urobilin  by  the  influence  of  sunlight  and  oxygen. 

In  preparing  urobilin  from  normal  urine,  precipitate  the  urine  with 
basic  lead  acetate  (Jaffe),  wash  the  precipitate  with  water,  dry  at  the 
ordinary  temperature,  then  boil  it  with  alcohol,  and  decompose  it  when 
cold  with  alcohol  containing  sulphuric  acid.  The  filtered  alcoholic  solution 
is  diluted  with  water,  saturated  with  ammonia,  and  then  treated  with  zinc- 
chloride  solution.  This  new  precipitate  is  washed  free  from  chlorine  with 
water,  boiled  with  alcohol,  dried,  dissolved  in  ammonia,  and  this  solution 
precipitated  writh  sugar  of  lead.  This  precipitate,  which  is  washed  with 
water  and  boiled  with  alcohol,  is  decomposed  by  alcohol  containing  sul- 
phuric acid,  the  filtered  alcoholic  solution  is  mixed  with  £  vol.  chloroform, 
diluted  with  water,  and  shaken  repeatedly,  but  not  too  energetically.  The 
urobilin  is  taken  up  by  the  chloroform.  This  last  is  washed  once  or  twice 
with  a  little  water  and  then  distilled,  leaving  the  urobilin.  The  pigment 
may  be  precipitated  directly  from  the  urine  rich  in  urobilin  by  ammonia 
and  zinc  chloride,  and  the  precipitate  treated  as  above  described  (Jaffe). 

1  SeeSalkowski,  Berlin,  klin.  Wochenschr. ,  1897,  and  Stokvis,  Zeitschr.  f.  Biologie,  34. 

2  Garrod  and  Hopkins,  Journ.  of  Physiol.,  20;  Saillet,  I.  c. 


UROBILIN.  519 

The  method  suggested  by  Mkiiy  (precipitation  with  ammonium  sul- 
phate) has  been  modified  by  Garrod  and  Hopkins  in  that  they  first  re- 
move the  uric  acid  by  saturating  with  ammonium  chloride  and  then  saturat- 
ing the  filtrate  with  ammonium  sulphate.  The  precipitated  urobilin  is 
thus  made  purer  than  by  saturating  with  the  sulphate  directly.  The 
urobilin  is  extActed  from  the  dried  precipitate  by  a  great  deal  of  water, 
reprecipitated  by  ammonium  sulphate,  and  this  procedure  repeated  several 
times  if  necessary.  The  dried  precipitate  finally  obtained  is  dissolved  in 
absolute  alcohol.  In  regard  to  small  details,  and  to  a  second  method  sug- 
gested by  these  experimenters,  avo  refer  to  the  original  work.1 

Saillet  extracts  the  urobilinogen  from  the  urine  by  shaking  with 
acetic  ether,  using  a  kerosene-oil  light.2 

The  color  of  the  acid  or  alkaline  solution,  the  beautiful  fluorescence  of 
the  ammoniacal  solution  treated  with  zinc  chloride,  and  the  absorption- 
bands  of  the  spectrum,  all  serve  as  means  of  detecting  urobilin.  In  fever- 
urines  the  urobilin  may  be  detected  directly  or  after  the  addition  of  ammo- 
nia and  zinc  chloride  by  its  spectrum.  It  may  also  sometimes  be  detected 
in  normal  urine,  either  directly  or  after  the  urine  has  stood  exposed  to 
the  air  until  the  chromogen  has  been  converted  into  urobilin.  If  it  cannot 
be  detected  by  means  of  the  spectroscope,  then  the  urine  may  be  treated 
with  a  mineral  acid  and  shaken  with  ether  or,  still  better,  with  amyl  alcohol. 
The  amyl-alcohol  solution  is,  either  directly  or  after  addition  of  a  strongly 
ammoniacal  alcoholic  solution  of  zinc  chloride,  tested  spectroscopically. 
According  to  Schlesinger  3  it  can  be  readily  detected  if  the  urine  is  pre- 
cipitated by  an  equal  volume  of  a  10  per  cent  solution  of  zinc  acetate  in 
absolute  alcohol.  Disturbing  bodies  are  here  precipitated  and  the  filtrate 
gives  the  fluorescence  directly  and  also  the  spectrum. 

In  the  quantitative  estimation  of  urobilin  we  proceed  as  follows, 
according  to  G.  Hoppe-Seyler:  4  100  c.  c.  of  the  urine  is  acidified  with 
sulphuric  acid  and  saturated  with  ammonium  sulphate.  The  precipitate 
is  collected  on  a  filter  after  some  time,  washed  with  a  saturated  solution  of 
ammonium  sulphate,  and  repeatedly  extracted  with  equal  parts  of  alcohol 
and  chloroform  after  pressing.  The  filtered  solution  is  treated  with  water 
in  a  separatory  funnel  until  the  chloroform  separates  well  and  becomes 
clear.  The  chloroform  solution  is  evaporated  on  the  water-bath  in  a 
weighed  beaker,  the  residue  dried  at  100°  C.,  and  then  extracted  with  ether. 
The  ethereal  extract  is  filtered,  the  residue  on  the  filter  dissolved  in  alcohol, 
and  transferred  to  the  beaker  and  evaporated,  then  dried  and  weighed. 
According  to  this  method  G.  Hoppe-Seyler  found  0.08-0.14  gram  of  urobilin 
in  one  day's  urine  of  a  healthy  person,  or  an  average  of  0.123  gram. 

t Urobilin  may  also  be  determined  spectro-photometricallv  according  to  Fr. 
Miller  or  to  Saillet.5  Saillet  found  that  the  limit  for  the  perceptibility 
of  the  absorption-bands  of  an  acid  urobilin  solution  lies  in  a  concentration  of 
1  milligram  of  urobilin  in  22  c.  c.  of  solution  when  the  thickness  of  the  layer  of 
fluid  is  15  mm.  In  a  quantitative  estimation  the  urobilin  solution  is  diluted  to 
this  limit  and  then  the  quantity  of  urobilin  calculated  from  the  extent  of  dilu- 

1  Journ.  of  Physiol. ,  20. 

J  In  regard  to  this  and  other  methods,  we  must  refer  the  reader  to  special  works. 

s  Deutsch.  med.  Wochenschr.,  1903. 

4  Virchow's  Arch.,  124. 

s  Fr.  Miiller,  see  Huppert-Xeubauer,  861 ;  Saillet,  1.  c. 


520  URINE. 

tion.  The  freshly  voided  urine,  shielded  from  light,  is  acidified  with  acetic  acid, 
completely  extracted  in  kerosene-oil  light  with  acetic  ether,  and  the  dissolved 
urobilinogen  oxidized  to  urobilin  with  nitric  acid.  On  the  addition  of  ammonia 
and  shaking  with  water  the  urobilin  passes  into  the  watery  solution.  This  is 
acidified  with  hydrochloric  acid  and  diluted  until  the  above  limit  is  reached. 

Uroerythrin  is  the  pigment  which  often  gives  the  beautiful  red  color  to 
the  urinary  sediments  (sedimentum  lateritium).  It  also  frequently  occurs, 
although  only  in  very  small  quantities,  dissolved  in  normal  urines.  The 
quantity  is  increased  after  great  muscular  activity,  after  profuse  perspira- 
tion, immoderate  eating,  or  partaking  of  alcoholic  drinks,  as  well  as  after 
digestive  disturbances,  fevers,  circulatory  disturbances  of  the  liver,  and  in 
many  other  pathological  conditions. 

Uroerythrin,  which  has  been  especially  studied  by  Zoja,  Riva,  and 
Garrod,1  has  a  pink  color,  is  amorphous,  and  is  very  quickly  destroyed  by 
light,  especially  when  in  solution.  The  best  solvent  is  amyl  alcohol;  acetic 
ether  is  not  so  good,  and  alcohol,  chloroform,  and  water  are  even  less  valu- 
able. The  very  dilute  solutions  show  a  pink  color;  but  on  greater  con- 
centration they  become  reddish  orange  or  fire-red.  They  do  not  fluoresce 
either  directly  or  after  the  addition  of  an  ammoniacal  solution  of  zinc  chlo- 
ride; but  they  have  a  strong  absorption,  beginning  in  the  middle  between 
D  and  E  and  extending  to  about  F,  and  consisting  of  two  bands  which 
are  connected  by  a  shadow  between  E  and  b.  Concentrated  sulphuric  acid 
colors  a  uroerythrin  solution  a  beautiful  carmine-red;  hydrochloric  acid 
gives  a  pink  color.  Alkalies  make  its  solutions  grass-green,  and  often  a 
play  of  colors  from  pink  to  purple  and  blue  is  observed. 

In  preparing  uroerythrin  from  the  sediment,  according  to  Garrod,  it  is  dis- 
solved in  water  at  a  gentle  heat  and  saturated  with  ammonium  chloride,  which 
precipitates  the  pigment  with  the  ammonium  urate.  This  is  purified  by  repeated 
solution  in  water  and  precipitation  with  ammonium  chloride  until  all  the  urobilin 
is  removed.  The  precipitate  is  finally  extracted  on  the  filter  in  the  dark  with 
warm  water,  filtered,  then  diluted  with  water,  any  hasmatoporphyrin  remaining 
is  removed  by  shaking  with  chloroform,  finally  faintly  acidified  with  acetic  acid 
and  shaken  with  chloroform,  which  takes  up  the  uroerythrin.  The  chloroform  is 
evaporated  in  the  dark  at  a  gentle  heat. 

Volatile  fatty  acids,  such  as  formic  acid,  acetic  acid,  and  perhaps  also  butyric 
acid,  occur  under  normal  conditions  in  human  urine  (v.  Jaksch),  also  in  that  of 
dogs  and  herbivora  (Schotten).  The  acids  poorest  in  carbon,  such  as  formic 
acid  and  acetic  acid,  are  more  constant  in  the  body  than  those  richer  in  carbon, 
and  therefore  the  relatively  greater  part  of  these  pass  unchanged  into  the  urine 
(SrnoTTEN).  No  mal  human  urine  contains  besides  these  bodies  others  which 
yield  acetic  acid  when  oxidized  by  potassium  dichromate  and  sulphuric  acid 
(v.  Jaksch).  The  quantity  of  volatile  fatty  acids  in  normal  urine  is,  according 
to  v.  Jaksch,  0.008-0.000  gram  per  twenty-four  hours,  and  according  to  v.  Roki- 
tansky,  0.054  gram.     The  quantity  is  increased  by  exclusive  farinaceous  food 


1  Zoja,  Arch.  Ital.  di.  clinica  med.,  1893,  and  Centralbl.  f.  d.  med.  Wissensch.,  1892; 
Riva,  Gaz.  med.  di  Torino,  Anno  43,  cited  from  Maly's  Jahresber.,  24;  Garrod,  Journ. 
of  Physiol.,  17  and  21. 


VARIOUS  ACIDS.     CARBOHYDRATE.  521 

(Rokitansky),  in  fever  and  in  certain  diseases,  while  in  others  it  is  dimin- 
ished (v.  Jaksch,  Rosenfelo).  Large  amounts  of  volatile  fatty  acids  are  pro- 
duced in  the  alkaline  fermentation  of  the  urine,  and  the  quantity  is  0-15  times 
as  large;  a.s  in  normal  urine  (8ALKOWBK]  ').  Non-volatile  fatty  acids  have  been 
detected  as  normal  constituents  of  urine  by  K.  Morner  and  Hyhbinette.2 

I'aralactic  Acid.  It  is  claimed  that  this  acid  occurs  in  the  urine  of  healthy 
persons  after  very  fatiguing  marches  (Colasanti  and  Moscatelli).  It  is  found 
in  larger  amounts  in  the  urine  in  acute  phosphorus-poisoning  or  acute  yellow 
atrophy  of  the  liver  (Schultzen  and  Riess).  According  to  the  investigations 
of  Hoppb-Sbtlbb,  Araki,  and  v.  Terrey  lactic  acid  passes  into  the  urine  as  soon 
as  the  supply  of  oxygen  is  decreased  in  any  way,  and  this  probably  explains  the 
occurrence  of  lactic  acid  in  the  urine  after  epileptic  attacks  (Inouye  and  Saiki). 
Minkowski  3  has  shown  that  lactic  acid  occurs  in  the  urine  in  large  quantities 
on  the  extirpation  of  the  liver  of  birds. 

Glycerophosphoric  acid  occurs  as  traces  in  the  urine,4  and  it  is  probably  a 
decomposition  product  of  lecithin.  The  occurrence  of  succinic  acid  in  normal 
urine  is  a  subject  of  discussion. 

Carbohydrates  and  Reducing  Substances  in  the  Urine.  The  occurrence 
of  dextrose  as  traces  in  normal  urine  is  highly  probable,  as  the  investiga- 
te mis  of  Brucke,  Abeles,  and  v.  Udranszky  show.  The  last  investiga- 
tor has  also  shown  the  habitual  occurrence  of  carbohydrates  in  the  urine, 
and  their  presence  has  been  positively  proved  by  the  investigations  of 
Baumann  and  Wedenski,  and  especially  by  Baisch.  Besides  dextrose 
normal  urine  contains,  according  to  Baisch,  another  not  well-studied 
variety  of  sugar;  according  to  Lemaire,  probably  isomaltose  is  present, 
and  besides  this  a  dextrin-like  carbohydrate  (animal  gum),  as  shown  by 
Landwehr,  Wedenski,  and  Baisch.  The  quantity  of  carbohydrates 
eliminated  under  normal  conditions  in  the  twenty-four  hours  urine  and 
determined  by  the  benzoylation  method,  which  is  perhaps  not  sufficiently 
trustworthy,  varies  considerably  between  1.5-5.09  grams.5 

Besides  traces  of  sugar  and  the  reducing  substances  previously  men- 
tioned, uric  acid  and  creatinine,  the  urine  contains  still  other  bodies  of  this 
character.  These  latter  are  partly  conjugated  compounds  of  glucuronic 
acid,  C6H10O7,  which  is  closely  allied  to  dextrose.  The  reducing  power  of 
normal  urine  corresponds,  according  to  various  investigators,  to  1.5-5.96 


'v.  Jaksch,  Zeitschr.  f.  physiol.  Chem.,  10;  Schotten,  ibid.,  7;  Rokitansky,  Wien. 
med.  Jahrbuch,  1887;  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  13;  Rosenfeld,  Deutsch, 
med.  Wochcnschr.,  29. 

3Skand.  Arch.  f.  Physiol.,  7. 

3  Colasanti  and  Moscatelli,  Moleschott's  Untersuch.,  14;  Schultzen  and  Reiss, 
Chem.  Centralbl.,  1869;  Araki,  Zeitschr.  f.  physiol.  Chem.,  15,  16,  17,  19.  See  also 
Irisawa,  ibid.,  17;  v.  Terrey,  Pfliiger's  Arch.,  65;  Schiitz,  Zeitschr.  f.  physiol.  Chem., 
19;  Inouye  and  Saiki,  ibid.,  38;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21  and  31. 

4  See  Pasqualis,  Maly's  Jahresber.,  24. 

5  Lemaire,  Zeitschr.  f.  physiol.  Chem.,  21;  Baisch,  ibid.,  18,  19,  and  20.  In  these 
as  well  as  in  Treupel,  ibid.,  16,  the  works  of  other  investigators  are  cited.  See  also 
v.  Alfthan,  Deutsch.  med.  Wochenschr.,  26. 


522  URINE. 

p.  m.  dextrose.1    That  portion  of  the  reduction  belonging  to  dextrose 
alone  is  equal  to  0.1-0.6  p.  m. 

Several  new  methods  for  the  determination  of  the  reducing  power  of  the  urine 
have  been  suggested.2 

Conjugated  glucuronates  occur,  as  indicated  by  Fluckiger  and  first 
positively  shown  by  Mayer  and  Neuberg,3  in  very  small  amounts  in  nor- 
mal urine.  They  occur  chiefly  as  phenol-  and  only  very  small  amounts 
of  indoxyl-  or  skatoxyl  glucuronates.  The  quantity  of  glucuronic  acid 
obtained  from  the  conjugated  glucuronates  is  estimated  as  0.04  p.  m.  by 
Mayer  and  Neuberg. 

Very  large  amounts  of  these  conjugated  glucuronates  occur  in  the  urine, 
on  the  other  hand,  after  partaking  of  various  therapeutic  agents  and  other 
substances,  such  as  chloral  hydrate,  camphor,  naphthol,  borneol,  turpen- 
tine, morphine,  etc.  According  to  P.  Mayer  as  stated  on  page  99,  in  the 
oxidation  of  dextrose,  a  part  of  it  forms  glucuronic  acid,  hence  it  is  to  be 
expected  that  the  glucuronic  acid  can  in  part  be  derived  from  the  dex- 
trose. As  a  conjugation  of  the  glucuronic  acid  with  other  bodies,  such  as 
aromatic  atomic  complexes,  prevents  the  combustion  of  this  acid  in  the 
animal  body,  it  ought  to  follow  that  after  the  introduction  of  such  an 
atomic  complex  in  the  body  during  a  glycosuria  that  a  corresponding 
reduction  of  the  glucose  elimination  would  take  place  with  the  increased 
excretion  of  conjugated  glucuronates.  In  order  to  prove  this  possibility 
O.  Loewi  4  fed  dogs  with  camphor  during  phlorhizin  diabetes  and  found 
that  the  above  expectation  was  not  true.  Although  large  quantities  of 
campho-glucuronic  acid  were  excreted,  the  sugar  excretion  was  only  slightly 
diminished  and  not  in  proportion  to  the  quantity  of  conjugated  glucuronate 
excreted,  which  tends  to  show  that  the  glucuronic  acid  is  not  produced 
from  the  dextrose,  neither  is  the  dextrose  the  mother-substance  of  this  acid. 

According  to  the  body  with  which  they  are  conjugated  the  glucuronates 
show  different  behavior;  they  all  rotate  the  plane  of  polarization  to  the 
left,  while  the  glucuronic  acid  itself  is  dextrorotatory.  On  taking  up 
water  they  split  into  glucuronic  acid  and  the  conjugated  group.  A  few 
reduce  copper  oxide  and  certain  other  metallic  oxides  in  alkaline  solution 
and  hence  cause  errors  in  the  investigation  of  the  urine  for  sugar.  As  the 
detection  of  conjugated  glucuronic  acids  is  connected  with  the  tests  for 
sugar  in  the  urine  we  will  treat  of  this  in  connection  with  these  tests. 


1  Pfliiger,  Zeitschr.  f.  physiol.  Chem.,  9.     See  also  Huppert-Neubauer,  72. 

2  See  Rosin,  Munch,  med.  Wochenschr. ,  46;  Niemilowicz,  Zeitschr.  f.  physiol. 
Chem.,  36;  Niemilowicz  with  Gittlemacher-Wilenko,  ibid.,  36,  and  Helier,  Compt.  rend., 
129. 

3  Fluckiger,  1.  c;  Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29. 

4  Arch.  f.  exp.  Path.  u.  Pharm.,  47. 


SULPHURIZED  COMPOUNDS.  523 

Organic  combinations  containing  sulphur  of  unknown  kind,  which  may 
in  small  part  consist  of  sidphocyanid.es,  0.04  (Gscheidlen)-O.H  p.  m. 
(I.  Munk  *),  cystin,  or  bodies  related  to  it,  taurin  derivatives,  chondroitin- 
sulphuric  acid,  and  protein  bodies,  but  in  greater  part  are  made  up  of  oxy- 
proteic  acid,  alloxyproteic  acid,  and  urojerric  acid,  are  found  in  human  as  well 
as  in  animal  urines.  The  sulphur  of  these  mostly  unknown  combinations  has 
been  called  "  neutral,"  to  differentiate  it  from  the  "  acid  "  sulphur  of  the  sul- 
phate and  ethereal-sulphuric  acids  (Salkowski  2).  The  neutral  sulphur  in 
normal  urine  as  determined  by  Salkowski  is  15  per  cent,  by  Stadthagen 
13.3-14.5  per  cent,  and  by  Lepixe  20  per  cent,  and  Harnack  and  Kleixe  3 
19-24  per  cent  of  the  total  sulphur.  In  starvation,  according  to  Fr. 
Miller,  with  insufficient  supply  of  oxygen  (Reale  and  Boeri,  Harnack 
and  Kleixe),  as  in  chloroform  narcosis  (Kast  and  Mester),  as  also  after 
the  introduction  of  sulphur  (Presch  and  Yvox  4),  the  quantity  of  neutral 
sulphur  is  increased.  The  quantity  of  neutral  sulphur  varies,  according 
to  Bexedikt  and  Freuxd,  within  rather  narrow  limits  and  is  dependent 
to  a  less  degree  than  the  sulphate  excretion  upon  the  extent  of  the  pro- 
teid  metabolism.  The  relationship  between  the  neutral  and  acid  sulphur 
depends  in  the  first  place  upon  the  extent  of  the  sulphuric-acid  excretion. 
According  to  Harnack  and  Kleixe,5  the  relationship  of  the  oxidized 
sulphur  to  the  total  sulphur  changes  always  in  the  same  way  as  the  relation- 
ship of  the  nitrogen  of  the  urea  to  the  total  nitrogen.  The  more  unoxidized 
sulphur  is  eliminated  the  more  abundant  is  the  nitrogen,  not  urea,  in  the 
urine,  a  statement  which  coincides  with  recent  observations  where  the 
neutral  sulphur  originates  chiefly  from  the  oxyproteic  acid,  the  alloxyproteic 
acid,  and  the  uroferric  acid. 

According  to  Lepixe,  a  part  of  the  neutral  sulphur  is  more  readily  oxidized 
(directly  with  chlorine  or  bromine)  into  sulphuric  acid  than  the  other  which  is  only 
converted  into  sulphuric  acid  after  fusing  with  potash  and  saltpeter.  According 
to  the  investigations  of  W.  Smith,8  it  is  probable  that  the  most  unoxidizable  part 
of  the  neutral  sulphur  occurs  as  sulpho-acids.  An  increased  elimination  of  neutral 
sulphur  has  been  observed  in  various  diseases,  such  as  pneumonia,  cystinuria, 
and  especially  where  the  flow  of  bile  into  the  intestine  is  prevented. 

The  total  quantity  of  sulphur  in  the  urine  is  determined  by  fusing  the  solid 
urinary  residue  with  saltpeter  and  caustic  alkali.  The  quantity  of  neutral  sulphur 
is  determined  as  the  difference  between  the  total  sulphur  and  the  sulphur  of  the 
sulphate  and  ethereal-sulphuric  acids.     The  readily  oxidizable  part  of  the  neutral 

1  Gscheidlen,  Pfliiger's  Arch.,  14;    Munk,  Virchow's  Arch.,  69. 

3  Ibid.,  oS,  and  Zeitschr.  f.  physiol.  Chem.,  9 

'Stadthagen,  Virchow's  Arch.,  100;  Lepine,  Compt.  rend.,  91  and  97;  Harnack 
and  Kleine,  Zeitschr.  f.  Biologic,  37. 

4  Fr.  Miiller,  Berl.  klin.  Wochenschr.,  1887;  Reale  and  Boeri,  Maly's  Jahrcsher.  24; 
Harnack  and  Kleine,  1.  c. ;  Presch,  Virchow's  Arch.,  119;  Yvon,  Arch,  de  Physiol. 
(5),  10. 

5  Benedikt,  Zeitschr.  f.  klin.  Med.,  36;  Harnack  and  Kleine,  1.  c. 
8  Lupine,  1.  c. ;  Smith,  Zeitschr.  f.  physiol.  Chem.,  17. 


524  URINE. 

sulphur  is  determined  by  oxidation  with  bromine  or  potassium  chlorate  and 
hydrochloric  acid  (Lepine,  Jerome  l). 

Sulphuretted  hydrogen  occurs  in  the  urine  only  under  abnormal  conditions 
or  as  a  decomposition  product.  This  conmound  may  be  produced  from  the 
neutral  sulphur  of  the  organic  substances  of  the  urine  by  the  action  of  certain 
bacteria  (Fr.  Muller,  Salkowski  2).  Other  investigators  have  given  hypo- 
sulphites as  the  source  of  the  sulphuretted  hydrogen.  The  occurrence  of  hypo- 
sulphites in  normal  human  urine,  which  is  asserted  by  Heffter,  is  disputed  by 
Salkowski  and  Presch.3  Hyposulphites  occur  constantly  in  cat's  urine  and, 
as  a  rule,  also  in  dog's  urine. 

Oxyproteic  acid  is  the  name  given  by  Bondzynski  and  Gottlieb  to  a 
nitrogenous  acid  containing  sulphur,  whose  existence  in  human  urine  was 
first  suggested  by  Topfer.  It  seems  to  be  a  normal  constituent  of  human 
and  dog 's  urine,  but  is  found  to  a  much  greater  extent  in  the  urine  of  dogs 
poisoned  with  phosphorus  (Bondzynski  and  Gottlieb).  According  to 
these  experimenters  it  has  the  formula  C43H82N14S031,  and  according  to 
Cloetta,4  who  calls  it  uroproteic  acid,  C66H116N20SO54.  It  does  not  contain 
any  loosely  combined  sulphur,  and  yields  no  tyrosin  on  cleavage.  It  does 
not  respond  either  to  the  xanthoproteic  or  the  biuret  reaction,  but  gives  a 
faint  test  with  Millon  's  reagent,  and  is  not  precipitated  by  phosphotungstic 
acid;  hence  on  this  account  it  leads  to  an  error  in  the  Pfluger  and  Boh- 
land  method  for  estimating  urea.  Its  barium  salt  is  soluble  in  water  but 
insoluble  in  alcohol,  and  serves  in  the  preparation  of  the  acid  from  the 
urine.  This  acid  is  precipitated  by  mercuric  acetate  or  nitrate,  but  not  by 
basic  lead  acetate.     It  gives  Ehrlich's  cliazo  reaction  (see  below). 

This  acid  is  considered  as  an  intermediary  oxidation  product  of  the 
proteids,  and  it  is  similar  in  certain  regards  to  Maly's  peroxyprotic  acid. 
The  amount  calculated  as  barium  salt,  according  to  Bondzynski  and 
Gottlieb,  may  be  3-4  grams,  and  somewhat  more,  according  to  Pregl,5 
and  its  nitrogen  amounts  to  about  2-3  per  cent  of  the  total  nitrogen. 

Alloxyproteic  acid  is  another  of  these  acids  isolated  by  Bondzynski  and 
Panek  6  from  the  urine.  The  formula  of  this  acid  has  not  been  determined. 
The  free  acid  contains  6  per  cent  sulphur.  The  barium  salt  contains 
28.76-32.05  per  cent  Ba,27  per  cent  C,  8.20-10.13  per  cent  N,  and  3.22-3.41 
per  cent  S.  Like  oxyproteic  acid  it  does  not  give  the  biuret  reaction 
and  is  not  precipitated  either  by  phosphotungstic  acid  or  by  tannic  acid 
or  by  potassium  ferrocyanide  and  acetic  acid.  It  differs  from  oxyproteic 
acid  by  a  different  behavior  of  its  salts,  and  also  by  the  fact  that  it  is  pre- 

1  Jerome,  Pfluger \s  Arch.,  00. 

2  Ft.  Muller,  Berlin,  klin.  Wochenschr. ,  1887;   Salkowski,  ibid.,  1888. 

5  Heffter,  Pfluger 's  Arch.,  38;  Salkowski,  ibid.,  39;  Presch,  Virchow's  Arch.,  119. 

*  Bondzynski  and  Gottlieb,  Centralbl.  f.  d.  med.  Wissensch.,  1897,  No  33;  Topfer, 
ibid.,  41;   Cloetta,  Arch.  f.  exp.  Path.  u.  Pharm.,  40. 

*  Pfluger 's  Arch.,  75. 

8  Ber.  d.  d.  chem.  Gesellsch.,  35. 


UROFERRIC  ACID.  525 

oipitated  by  basic  load  acetate  and  by  not  giving  Ehrlich's  diaao  reaction. 

The  daily  amount  of  this  acid  II  wimakstex  calculates  as  1.2  grams,  equal 
to  O.GS  per  cent  of  the  total  nitrogen. 

The  two  acids  are  prepared  from  the  urine  by  precipitating  it  with  barium 
hydrate  and  calcium  hydrate,  removing  the  excess  with  carbon  dioxide, 
and  then  evaporating  to  a  syrup.  After  treating  this  syrup  with  alcohol- 
ether  the  residue  is  dissolved  in  water  and  then  precipitated  in  acetic-acid 
solution  with  mercuric  acetate.  The  precipitate,  which  contains  both 
acids,  is  treated  with  II,S,  then  the  calcium  salts  prepared  and  their  solu- 
tion precipitated  with  basic  lead  acetate,  which  only  precipitates  the 
alloxyproteic  acid.  Finally  the  barium  or  silver  salt  of  each  acid  is  pre- 
pared separately. 

Uroferric  add  is  an  acid  isolated  by  Thiele  l  from  the  urine,  according 
to  Siegfried's  method  for  preparing  pure  peptone.  It  also  contains 
sulphur,  3.46  per  cent,  and  has  the  formula  C^HjgXgSO^.  The  acid  forms 
a  white  powder  which  is  readily  soluble  in  water,  saturated  ammonium- 
sulphate  solution,  and  methyl  alcohol.  It  is  soluble  with  difficulty  in 
absolute  alcohol,  insoluble  in  benzene,  chloroform,  ether,  and  acetic 
ether.  About  one  half  of  the  sulphur  can  be  split  off  as  sulphuric  acid 
on  boiling  with  hydrochloric  acid.  The  acid  gives  neither  the  biuret 
test  nor  Millox's  or  Adamkiewicz's  reactions.  It  is  precipitated  by 
mercuric  nitrate  and  sulphate  and  also  by  phosphotungstic  acid  (differing 
from  the  other  two  above-mentioned  acids).  This  acid  is  hexabasic  and 
its  specific  rotation  is  (a)l,8  =  —  32.5°.  On  cleavage  it  yields  melanine 
substances,  sulphuric  acid,  aspartic  acid,  but  no  hexon  bases. 

Organic  combinations  containing  phosphorus  (glycerophosphoric  acid,  phospho- 
carnic  acid  (Rock wood),  etc.),  which  yield  phosphoric  acid  on  fusing  with  salt- 
peter and  caustic  alkali,  are  also  found  in  urine  (Lepixe  and  Eymoxxet,  Oertel  2). 
With  a  total  elimination  of  about  2.0  grams  total  P205,  Oertel  found  on  an 
average  about  O.Oo  gram  P205  as  phosphorus  in  organic  combination. 

Enzymes  of  various  kinds  have  been  isolated  from  the  urine.  Among  these 
may  be  mentioned  pepsin  (Brucke  and  othi-rs),  which,  according  to  Matthes, 
undoubtedly  originates  from  the  stomach,  and  a  diastolic  enzyme  (Cohnheim  and 
others).     The  occurrence  of  rennin  and  trypsin  in  the  urine  is  doubtful.3 

Mucin.  The  nubecula  consists,  as  shown  by  K.  Morxer,4  of  a  mucoid  which 
contains  12.74  per  cent  X  and  2.3  per  cent  S.  This  mucoid,  which  apparently 
originates  in  the  urinary  passages,  may  pass  to  a  slight  extent  into  solution  in  the 
urine.  In  regard  to  the  nature  of  the  mucins  and  nucleoalbumins  otherwise 
occurring  in  the  urine  we  refer  the  reader  to  the  pathological  constituents  of  the 
urine. 

1  Zeitschr.  f.  physiol.  Chem.,  37. 

7  Rockwood,  Du  Bois-Reymond 's  Arch.,  1895;  Oertel,  Zeitschr.  f.  physiol.  Chem., 
26,  which  cites  the  other  works.  See  also  Keller,  Zeitschr.  f.  physiol.  Chem.,  29j 
Mandel  and  Oertel,  X.  Y.  Univ.  Bull.  Med.  Sciences,  1. 

3  In  regard  to  the  literature  on  enzymes  in  the  urine,  see  Huppert-Neubauer,  599; 
Matthes,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 

*Skand.  Arch.  f.  Physiol.,  6. 


526  URINE. 

Ptomaines  and  leucomaines,  or  poisonous  substances  of  an  unknown  kind, 
which  are  often  described  as  alkaloidal  substances,  occur  in  normal  urine  (Pouchet,, 
Bouchard,  Aducco,  and  others).  Under  pathological  conditions  the  quantity 
of  these  substances  may  be  increased  (Bouchard,  Lepine  and  Guerin,  Villiers, 
Griffiths,  Albu,  and  others).  Within  the  last  few  years  the  poisonous  proper- 
ties of  urine  have  been  the  subject  of  more  thorough  investigation,  especially  by 
Bouchard.  He  found  that  the  night  urine  is  less  poisonous  than  the  day  urine, 
and  that  the  poisonous  constituents  of  the  day  and  night  urines  have  not  the  same 
action.  In  order  to  be  able  to  compare  the  toxic  power  of  the  urine  under  different 
conditions,  Bouchard  determines  the  urotoxic  coefficient,  which  is  the  weight 
of  rabbit  in  kilos  that  is  killed  by  the  quantity  of  urine  excreted  in  twenty-four 
hours  by  1  kilo  of  the  person  experimented  upon.1 

Baumann  and  v.  Udranszky  have  shown  that  ptomaines  may  occur  in  the 
urine  under  pathological  conditions.  They  demonstrated  the  presence  of  the  two 
ptomaines  discovered  and  first  isolated  by  Brieger — putrescin,  C4H12N2  (tetra- 
methylendiamine) ,  and  cadaverin,  C5H14N2  (pentamethylendiamine) — in  the  urine 
of  a  patient  suffering  from  cystinuria  and  catarrh  of  the  bladder.  Cadaverin 
has  later  been  found  by  Stadthagen  and  Brieger  in  the  urine  in  two  cases  of 
cystinuria.  Brieger,  v.  Udranszky  and  Baumann,  and  Stadthagen  have 
shown  that  neither  these  nor  other  diamines  occurs  under  physiological  conditions, 
while  Dombrowski,2  on  the  contrary,  found  cadaverin  besides  another  ptomaine 
with  the  formula  C2H15N02,  and  mannite  in  normal  urines.  The  occurrence  in 
normal  urine  of  any  ' '  urine  poison ' '  is  denied  by  certain  investigators,  such  as 
Stadthagen,  Beck,  and  v.  d.  Bergh.3  The  poisonous  action  of  the  urine, 
according  to  them,  is  due  in  part  to  the  potassium  salts  and  in  part  to  the  sum 
of  the  toxicity  of  the  other  normal  urinary  constituents  (urea,  creatinine,  etc.), 
which  have  very  little  poisonous  action  individually.  The  same  experimenters 
have  presented  very  forcible  objections  to  Bouchard's  doctrine. 

Many  substances  have  been  observed  in  animal  urine  which  are  not  found  in 
human  urine.  To  these  belong  the  above-described  kynurenic  acid,  urocanic  acid, 
also  found  in  dog's  urine  and  which  seems  to  stand  in  some  relationship  to  the 
purin  bases;  damaluric  acid  and  damolic  acid  (according  to  Schotten,4  probably 
a  mixture  of  benzoic  acid  with  volatile  fatty  acids),  obtained  by  the  distillation 
of  cow's  urine;  and  lastly,  lithuric  acid,  found  in  the  urinary  concrements  of  cer- 
tain animals. 

III.   Inorganic  Constituents  of  Urine. 

Chlorides.  The  chlorine  occurring  in  the  urine  is  undoubtedly  combined 
with  the  bases  contained  in  this  excretion;  the  chief  part  is  in  combination 
with  sodium.  In  accordance  with  this,  the  quantity  of  chlorine  in  the 
urine  is  generally  expressed  as  NaCl. 

The  question  as  to  whether  a  part  of  the  chlorine  contained  in  the  urine 
exists  as  organic  combinations,  as  considered  by  Berlioz  and  Lepinois,  is 
still  disputed.5 

1  A  complete  bibliography  on  the  ptomaines  and  leucomaines  of  the  urine  is  found 
in  Huppert-Neubauer,  403. 

2  Baumann  and  Udranszky,  Zeitschr.  f.  physiol.  Chem.,  13;  Stadthagen  and  Brieger, 
Virchow's  Arch.,  115;  Dombrowski,  Arch,  polonais.  d.  sciences  biol.,  1903. 

3  Stadthagen,  Zeitschr.  f.  klin.  Med.,  15,  Beck,  Pfliigej's  Arch.,  71;  v.  d.  Bergh, 
Zeitschr.  f.  klin.  Med.,  35. 

*  Zeitschr.  f.  physiol.  Chem.,  7. 

5  Berlioz  and  Lepinois,  see  Chem.  Centralbl.,  1894,  1,  and  1895,  1;  also  Petit  and 
Terrat,  ibid.,  1894,2,  and  Vitali,  ibid.,  1897,  2;  Viele  et  Moitessier,  Maly's  Jahresber., 
31;  Meillere,  ibid. ;   Bruno,  ibid.,  452. 


CHLORIDES.  5_>7 

The  quantity  of  chlorine  combinations  in  the  urine  is  subject  to  consider- 
able variation.  In  general  the  quantity  from  a  healthy  adult  on  a  mixed 
diet  is  10- 15  prams  of  XaCl  per  twenty-four  hours.  The  quantity  of  common 
salt  in  the  urine  depends  chiefly  upon  the  quantity  of  salt  in  the  food,  with 
which  the  elimination  of  chlorine  increases  and  decreases.  The  free  drink- 
ing of  water  also  increases  the  elimination  of  chlorine,  which  is  greater 
during  activity  than  during  rest  (at  night).  Certain  organic  chlorine  com- 
binations, such  as  chloroform,  may  increase  the  elimination  of  inorganic 
chlorides  by  the  urine  (Zeller,  Kast  l). 

In  diarrhoea,  in  quick  formation  of  large  transudates  and  exudates,  also 
in  specially  marked  cases  of  acute  febrile  diseases  at  the  time  of  the  crisis, 
the  elimination  of  XaCl  is  materially  decreased.  The  excretion  of  chlorine 
may  vary  considerably  in  disease,  but  still  the  XaCl  taken  with  the  food 
has  here,  as  in  physiological  conditions,  a  great  influence  on  the  NaCl 
excretion. 

The  quantitative  estimation  of  chlorine  in  the  urine  is  most  simply  per- 
formed by  titration  with  silver-nitrate  solution.  The  urine  must  not 
contain  either  proteid  (which  if  present  must  be  removed  by  coagulation) 
or  iodine  or  bromine  compounds. 

In  the  presence  of  bromides  or  iodides  evaporate  a  measured  quantity  of  the 
urine  to  dryness,  fuse  the  residue  with  saltpeter  and  soda,  dissolve  the  fused  mass 
in  water,  and  remove  the  iodine  or  bromine  by  the  addition  of  dilute  sulphuric 
acid  and  some  nitrite,  and  thoroughly  shake  with  carbon  disulphide.  The  liquid 
thus  obtained  may  now  be  titrated  with  silver  nitrate  according  to  Volhabd's 
method.  The  quantity  of  bromide  or  iodide  is  calculated  as  the  difference  be- 
tween the  quantity  of  silver-nitrate  solution  used  for  the  titration  of  the  solution 
of  the  fused  mass  and  the  quantity  used  for  the  corresponding  volume  of  the 
original  urine. 

The  otherwise  excellent  titration  method  of  Mohr,  according  to  which 
we  titrate  with  silver  nitrate  in  neutral  liquids,  using  neutral  potassium 
chromate  as  an  indicator,  cannot  be  used  directly  on  the  urine  in  careful 
work.  Organic  urinary  constituents  are  also  precipitated  by  the  silver  salt, 
and  the  results  are  therefore  somewhat  high  for  the  chlorine.  If  this  met  In  m  I 
is  to  be  employed,  the  organic  urinary  constituents  must  first  be  destroyed. 
For  this  purpose  evaporate  to  dryness  5-10  c.  c.  of  the  urine,  after  the 
addition  of  1  gram  of  chlorine-free  soda  and  1-2  grams  chlorine-free  salt- 
peter, and  carefully  fuse.  The  mass  is  dissolved  in  water,  acidified  faintly 
with  nitric  acid,  and  then  neutralized  exactly  with  pure  calcium  carbonate. 
This  neutral  solution  is  used  for  the  titration. 

The  silver-nitrate  solution  may  be  a  X/10  one.  It  is  often  made  of 
such  a  strength  that  each  cubic  centimeter  corresponds  to  0.000  gram  CI  or 
0.01  gram  XaCl.  This  last-mentioned  solution  contains  29.075  grams  of 
AgNOj  in  1  liter. 


'Zeller,  Zeitschr.  f.  physiol.  Chem.,  S;    Kast,  ibid.,  11;    Vitali,  Chem.   Centralbl.. 
S99,  2. 


528  URINE. 

Fretjxd  and  Toepfer,  as  well  as  Bodtker,1  have  suggested  modifica- 
tions of  Mohr's  method. 

Volhard's  Method.  Instead  of  the  preceding  determination,  Vol- 
hard's  method,  which  can  be  performed  directly  on  the  urine,  may  be 
employed.  The  principle  is  as  follows:  All  the  chlorine  from  the  urine 
acidified  with  nitric  acid  is-  precipitated  by  an  excess  of  silver  nitrate, 
filtered,  and  in  a  measured  part  of  the  filtrate  the  quantity  of  silver  added 
in  excess  is  determined  by  means  of  a  sulphocyanide  solution.  This  excess 
of  silver  is  completely  precipitated  by  the  sulphocyanide,  and  a  solution  of 
some  ferric  salt,  which,  as  is  well  known,  gives  a  blood-red  reaction  with 
the  smallest  quantity  of  sulphocyanide,  is  used  as  an  indicator. 

We  require  the  following  solutions  for  this  titration:  1.  A  silver- 
nitrate  solution  which  contains  29.075  grams  of  AgN03  per  liter  and  of  which 
each  cubic  centimeter  corresponds  to  0.01  gram  NaCl  or  0.00607  gram  CI. 
2.  A  saturated  solution  at  the  ordinary  temperature  of  chlorine-free  iron 
alum  or  ferric  sulphate.  3.  Chlorine-free  nitric  acid  of  a  specific  gravity 
of  1.2.  4.  A  potassium-sulphocyanide  solution  which  contains  8.3  grams 
KCNS  per  liter,  and  of  which  2  c.  c.  corresponds  to  1  c.  c.  of  the  silver- 
nitrate  solution. 

About  9  grams  of  potassium  sulphocyanide  are  dissolved  in  water  and  diluted 
to  1  liter.  The  quantity  of  KCNS  contained  in  this  solution  is  determined  by  the 
silver-nitrate  solution  in  the  following  Avay:  Measure  exactly  10  c.  c.  of  the  silver 
solution  and  treat  it  with  5  c.  c.  of  nitric  acid  and  1-2  c.  c.  of  the  ferric-salt  solu- 
tion and  dilute  with  water  to  about  100  c.  c.  Now  the  sulphocyanide  solution 
is  added  from  a  burette,  constantly  stirring  until  a  permanent  faint-red  colora- 
tion of  the  liquid  takes  place.  The  quantity  of  sulphocyanide  found  in  the  solu- 
tion by  this  means  indicates  how  much  it  must  be  diluted  to  be  of  the  proper 
strength.  Titrate  once  more  with  10  c.  c.  of  AgN03  solution  and  correct  the  sul- 
phocyanide solution  by  the  careful  addition  of  water  until  20  c.  c.  exactly  corre- 
sponds to  10  c.  c.  of  the  silver  solution. 

The  determination  of  the  chlorine  in  the  urine  is  performed  by  this 
method  in  the  following  way:  Exactly  10  c.  c.  of  the  urine  is  placed  in  a 
flask  which  has  a  mark  corresponding  to  100  c.  c.  and  which  is  provided 
with  a  stopper;  5  c.  c.  of  nitric  acid  is  added;  dilute  with  about  50  c.  c.  of 
wrater  and  then  allow  exactly  20  c.  c.  of  the  silver-nitrate  solution  to  flow 
in.  Close  the  flask  with  the  stopper  and  shake  well,  remove  the  stopper 
and  wash  it  with  distilled  water  into  the  flask,  and  fill  the  flask  to 
the  100-c.  c.  mark  with  distilled  water.  Close  again  with  the  stopper, 
carefully  mix  by  shaking,  and  filter  through  a  dry  filter.  Measure  off 
50  c.  c.  of  the  filtrate  by  means  of  a  dry  pipette,  add  3  c.  c.  of  ferric- 
salt  solution,  and  allow  the  sulphocyanide  solution  to  flow  in  until  the 
liquid  above  the  precipitate  has  a  permanent  red  color.  The  calculation 
is  very  simple.  For  example,  if  4.6  c.  c.  of  the  sulphocyanide  solu- 
tion was  necessary  to  produce  the  final  reaction,  then  for  100  c.  c.  of  the 
filtrate  (  =  10  c.  c.  urine)  9.2  c.  c.  of  this  solution  is  necessary.  9.2  c.  c.  of 
the  sulphocyanide  solution  corresponds  to  4.6  c.  c.  of  the  silver  solution, 
and  since  20—4.6=15.4  c.  c.  of  the  silver  solution  was  necessary  to  com- 
pletely precipitate  the  chlorides  in  10  c.  c.  of  the  urine,  then  10  c.  c.  con- 
tains 0.154  gram  of  NaCl.     The  quantity  of  sodium  chloride  in  the  urine 

1  Freund  and  Toepfer,  see  Maly's  Jahresber.,  22;  Bodtker,  Zeitschr.  f.  physiol. 
Chem.,  20. 


PIIOSPIIA  TES.  529 

is  therefore  1.64  per  cent,  or  15. 1  p.  in.  Jf  we  always  use  10  c.  c.  for  the 
determination,  and  always  20  c.  c.  of  AgNOj  solution,  and  dilute  with  water 
to  100  c.  c,  the  quantity  of  NaCl  in  1000  parts  of  the  urine  is  found  by  sub- 
tracting from  20  the  number  of  cubic  centimeters  of  sulphocyanide  (R)  re- 
quired with   50  C.  C.  of  the  filtrate.     The  quantity  of  NaCl  p.  m.  therefore 

under  these  circumstances  =  20—  R,  and  the  percentage  of  NaCl  =  — ^ — . 

The  approximate  estimation  of  chlorine  in  the  urine  (which  must  be 
free  from  proteid)  is  made  by  strongly  acidifying  with  nitric  acid  and  then 
adding  to  it,  drop  by  drop,  a  concentrated  silver-nitrate  solution  (1:8). 
In  a  normal  quantity  of  chlorides  the  drop  sinks  to  the  bottom  as  a  rather 
compact  cheesy  lump.  In  diminished  quantities  of  chlorides  the  precipi- 
tate is  less  compact  and  coherent,  and  in  the  presence  of  very  little  chlorine 
a  fine  white  precipitate  or  only  a  cloudiness  or  opalescence  is  obtained. 

Phosphates.  Phosphoric  acid  occurs  in  acid  urines  partly  as  dihydrogen, 
MH,P(V  and  partly  as  monohydrogen,  M2HP04,  phosphates,  both  of 
which  may  be  found  in  acid  urines  at  the  same  time.  Ott  l  found  that  on 
an  average  60  per  cent  of  the  total  phosphoric  acid  was  di-  and  40  per 
cent  was  monohydrogen  phosphate.  The  total  quantity  of  phosphoric  acid 
is  very  variable  and  depends  on  the  character  and  the  quantity  of  food.  The 
average  quantity  of  P205  is  in  round  numbers  2.5  grams,  with  a  variation  of 
1-5  grams  per  day.  A  small  part  of  the  phosphoric  acid  of  the  urine  orig- 
inates from  the  burning  of  organic  compounds,  such  as  nuclein,  protagon,  and 
lecithin,  within  the  organism;  on  exclusive  feeding  with  substances  rich  in 
nuclein  or  pseudonuclein  the  quantity  of  phosphates  is  essentially  increased; 
still  it  is  undecided  to  what  extent  the  excretion  of  phosphoric  acid  is  a 
measure  of  the  absorption  and  decomposition  of  these  bodies.2  The  greater 
part  originates  from  the  phosphates  of  the  food,  and  the  quantity  of  phos- 
phoric acid  eliminated  is  greater  when  the  food  is  rich  in  alkali  phosphates 
in  proportion  to  the  quantity  of  lime  and  magnesium  phosphates.  If 
the  food  contains  much  lime  and  magnesia,  large  quantities  of  earthy 
phosphates  are  eliminated  by  the  excrement;  and  even  though  the 
food  contains  considerable  amounts  of  phosphoric  acid  in  these  cases,  the 
quantity  excreted  by  the  urine  is  small.  This  is  true  especially  for 
herbivora,  in  which  the  kidneys  are  the  chief  organs  for  the  excretion 
of  alkali  phosphates.  In  man,  according  to  Ehrstrom,  the  content  of 
lime  in  the  food  seems  to  play  no  important  r61e,  as  in  his  experiments 
about  one  half  of  the  phosphoric  acid  taken  as  CaHP04  was  absorbed; 
still  the  extent  of  phosphoric-acid  excretion  through  the  urine  depends 
in  man  not  only  upon  the  total  quantity  of  phosphoric  acid  in  the  food  but 

1  Zeitschr.  f.  physiol.  Chem.,  10. 

2  See  A.  Gumlich,  Zeitschr.  f.  physiol.  Chem.,  18;  Roos,  ibid.,  21;  Wointraud, 
Du  Bois-Reymond 's  Arch.,  1895;  Milroy  and  Malcolm,  Journ.  of  Physiol.,  23;  Roh- 
mann  and  Steinitz,  Pfliiger's  Arch.,  72;  Loewi,  Arch,  f  exp.  Path.  u.  Pharm.,  44 
and  45. 


530  URINE. 

also  upon  the  relative  amounts  of  the  alkaline  earths  and  the  alkali  salts  of 
the  food.  In  carnivora,  in  which  phosphate  injected  subcutaneously  is 
eliminated  by  the  intestine  (Bergmann),  the  urine  is  habitually  poor  in 
phosphates.1 

As  the  extent  of  the  elimination  of  phosphoric  acid  is  mostly  dependent 
upon  the  character  of  the  food  and  the  absorption  of  the  phosphates  in  the 
intestine,  it  is  apparent  that  the  relationship  between  the  nitrogen  and 
phosphoric-acid  excretion  cannot  run  parallel.  This  is  in  fact  so,  and,, 
according  to  Ehrstrom,  the  organism  has  the  power  of  accumulating  large 
quantities  of  phosphorus  for  a  relatively  long  time  independent  of  the 
condition  of  the  nitrogen  balance.  With  a  certain  regular  food  the  rela- 
tionship between  nitrogen  and  phosphoric  acid  in  the  urine  can  be  kept 
nearly  constant.  Thus  on  feeding  with  an  exclusive  meat  diet,  as  ob- 
served by  Voit2  in  dogs,  when  the  nitrogen  and  phosphoric  acid  (P205) 
of  the  food  exactly  reappeared  in  the  urine  and  faeces,  the  relationship  was 
8.1:1.  In  starvation  this  relationship  is  changed,  namely,  relatively  more 
phosphoric  acid  is  eliminated,  which  seems  to  indicate  that  besides  flesh 
and  related  tissues,  another  tissue  rich  in  phosphorus  is  largely  destroyed. 
The  starvation  experiments  show  that  this  is  the  bone-tissue.  According 
to  Preysz,  Olsavszky,  Klug,  and  I.  Munk  3  the  elimination  of  phosphoric 
acid  is  considerably  increased  by  intense  muscular  work. 

As  the  phosphoric  acid  is  in  part  derived  from  the  nucleins  it  would  be 
expected  that  in  those  diseases  in  which  the  excretion  of  alloxuric  bodies 
was  increased  the  phosphoric  acid  would  also  be  augmented.  This  is  not 
the  case,  and  indeed  we  have  observed  cases  with  an  increased  elimination 
of  alloxuric  bodies  with  a  diminution  in  the  phosphoric-acid  excretion. 
Cases  of  leucaemia  have  been  observed  in  which  the  phosphoric-acid  excre- 
tion was  reduced,  although  there  was  a  pronounced  increase  in  the  number 
of  leucocytes.  In  these  cases  there  may  be  a  subsequent  excretion  or 
retention  of  phosphoric  acid.  This  last  condition  occurs  also  in  inflamma- 
tory and  renal  diseases.  The  urine  sometimes  has  the  tendency  of  pre- 
cipitating the  earthy  phosphates  either  spontaneously  or  after  warming, 
and  this  has  been  called  phosphaturia.  We  are  dealing  here,  it  seems,  with 
a  diminished  excretion  of  phosphoric  acid  and  an  increased  elimination  of 
lime,  or  at  least  an  essentially  different  relationship  between  the  phosphoric 
acid  and  the  alkaline  earths  of  the  urine,  as  compared  to  the  normal 
(Paxek,  Iwaxoff,  Soetber  and  Krieger  4). 

1  Ehrstrom,  Skand.  Arch.  f.  Physiol.,  14;  Bergmann,  Arch.  f.  exp.  Path.  u.  Pharm.,  47. 

2  Physiologie  des  allgemeinen  Stoffwechsels  und  der  Erniihrung  in  L.  Hermann's 
Handbuch,  6,  Thl.  1,  79. 

8  Preysz,  see  Maly's  Jahresber.,  21;  Olsavszky  and  Klug,  Pfluger's  Arch.,  54; 
Munk,  Du  Bois-Reymond's  Arch.,  1895. 

4Panek,  see  Maly's  Jahresber.,  30,  112;  Iwanoff,  Biochem.  Centralbl.,  1,  710; 
Soetber  and  Krieger,  Deutsch.  Arch.  f.  klin.  Med.,  72. 


ESTIMATION  OF  PHOSPHORIC  ACID.  531 

Quantitative  Estimation  of  the  Total  Phosphoric  Acid  in  the  Urine.  This 
estimation  is  most  simply  performed  by  titrating  with  a  solution  of  ura- 
nium acetate.  The  principle  of  the  titration  is  as  follows:  A  warm  solu- 
tion of  phosphates  containing  free  acetic  acid  gives  a  whitish-yellow  pre- 
cipitate of  uranium  phosphate  with  a  solution  of  a  uranium  salt.  This 
precipitate  is  insoluble  in  acetic  acid,  but  dissolves  in  mineral  acids,  and 
on  this  account  there  is  always  added,  in  titrating,  a  certain  quantity  of 
sodium-acetate  solution.  Potassium  ferrocyanide  is  used  as  the  indicator, 
which  does  not  act  on  the  uranium-phosphate  precipitate,  but  gh 
reddish-brown  precipitate  or  coloration  in  the  presence  of  the  smallest 
amount  of  soluble  uranium  salt.  The  solutions  necessary  for  the  titration 
are:  1.  A  solution  of  a  uranium  salt  of  which  each  cubic  centimeter  cor- 
responds to  0.005  gram  P205  and  which  contains  20.3  grams  of  uranium 
oxide  per  liter.  20  c.  c.  of  this  solution  corresponds  to  0.100  gram  P205. 
2.  A  solution  of  sodium  acetate.  3.  A  freshly  prepared  solution  of  potas- 
sium ferrocyanide. 

The  uranium  solution  is  prepared  from  uranium  nitrate  or  acetate.  Dissolve 
about  35  grams  uranium  acetate  in  water,  add  some  acetic  acid  to  facilitate  solu- 
tion, and  dilute  to  1  liter.  The  strength  of  this  solution  is  determined  by  titrating 
with  a  solution  of  sodium  phosphate  of  known  strength  (10.085  grams  crystallized 
salt  in  1  liter,  which  corresponds  to  0.100  gram  P,05  in  50  c.  c. ).  Proceed  in  the 
same  way  as  in  the  titration  of  the  urine  (see  below),  and  correct  the  solution  by 
diluting  with  water,  and  titrate  again  until  20  c.  c.  of  the  uranium  solution  corre- 
sponds exactly  to  50  c.  c.  of  the  above  phosphate  solution. 

The  sodium-acetate  solution  should  contain  10  grams  sodium  acetate  and 
10  grams  cone,  acetic  acid  in  100  c.  c.  For  each  titration  5  c.  c.  of  this  solution 
is  used  with  50  c.  c.  of  the  urine. 

In  performing  the  titration,  mix  50  c.  c.  of  filtered  urine  in  a  beaker 
with  5  c.  c.  of  the  sodium  acetate,  cover  the  beaker  with  a  watch-glass,  and 
warm  over  the  water-bath.  Then  allow  the  uranium  solution  to  flow  in 
from  a  burette,  and  when  the  precipitate  does  not  seem  to  increase,  place 
a  drop  of  the  mixture  on  a  porcelain  plate  with  a  drop  of  the  potassium- 
ferrocyanide  solution.  If  the  amount  of  uranium  solution  added  has  not  been 
sufficient,  the  color  will  remain  pale  yellow  and  more  uranium  solution  must 
be  added;  but  as  soon  as  the  slightest  excess  of  uranium  solution  has  been 
used  the  color  becomes  faint  reddish  brown.  When  this  point  has  been 
obtained,  warm  the  solution  again,  and  add  another  drop.  If  the  color 
remains  of  the  same  intensity,  the  titration  is  ended;  but  if  the  color  varies, 
add  more  uranium  solution,  drop  by  drop,  until  a  permanent  coloration  is 
obtained  after  warming,  and  now  repeat  the  test  with  another  50  c.  c.  of 
the  urine.  The  calculation  is  so  simple  that  it  is  unnecessary  to  give  an 
example. 

In  the  above  manner  one  determines  the  total  quantity  of  phosphoric 
acid  in  the  urine.  If  we  wish  to  know  the  phosphoric  acid  combined  with 
alkaline  earths  or  with  alkalies,  we  first  determine  the  total  phosphoric 
acid  in  a  portion  of  the  urine  and  then  remove  the  earthy  phosphates  in 
another  portion  by  ammonia.  The  precipitate  is  collected  on  a  filter, 
washed,  transferred  into  a  beaker  with  water,  treated  with  acetic  acid,  and 
dissolved  by  warming.  This  solution  is  now  diluted  to  50  c.  c.  with  water, 
and  5  c.  c.  sodium-acetate  solution  added,  and  titrated  with  uranium  solu- 
tion.   The  difference  between  the  two  determinations  gives  the  quantity 


532  URINE. 

of  phosphoric  acid  combined  with  the  alkalies.  The  results  obtained  are 
not  quite  accurate,  as  a  partial  transformation  of  the  monophosphates  of 
the  alkaline  earths  and  also  calcium  diphosphate  into  triphosphates  of  the 
alkaline  earths  and  ammonium  phosphate  takes  place  on  precipitating  with 
ammonia,  and  the  method  gives  too  high  results  for  the  phosphoric  acid 
combined  with  alkalies  and  remaining  in  solution. 

Sulphates.  The  sulphuric  acid  of  the  urine  originates  only  to  a  very- 
small  extent  from  the  sulphates  of  the  food.  A  disproportionately  greater 
part  is  formed  by  the  burning  within  the  body  of  the  proteids  which  contain 
sulphur,  and  it  is  chiefly  this  formation  of  sulphuric  acid  from  the  proteids 
which  gives  rise  to  the  previously  mentioned  excess  of  acids  over  the  bases 
in  the  urine.  The  quantity  of  sulphuric  acid  eliminated  by  the  urine 
amounts  to  about  2.5  grams  H2S04  per  day.  As  the  sulphuric  acid  chiefly 
originates  from  the  proteids,  it  follows  that  the  elimination  of  sulphuric 
acid  and  the  elimination  of  nitrogen  runs  nearly  parallel,  and  the  relation- 
ship N:H2S04  is  about  5:1.  A  complete  parallelism  can  hardly  be  ex- 
pected, as  in  the  first  place  a  part  of  the  sulphur  is  always  eliminated  as 
neutral  sulphur,  and  secondly,  because  the  small  proportion  of  sulphur  in 
different  proteid  bodies  undergoes  greater  variation  as  compared  with  the 
large  proportion  of  nitrogen  contained  therein.  In  general  the  relationship 
between  the  elimination  of  nitrogen  and  sulphuric  acid  under  normal  and 
under  diseased  conditions  runs  rather  parallel.  Sulphuric  acid  occurs  in  the 
urine  partly  preformed  (sulphate-sulphuric  acid)  and  partly  as  ethereal- 
sulphuric  acid.  The  first  is  designated  as  A-  and  the  other  as  5-sulphuric 
acid. 

The  quantity  of  total  sulphuric  acid  is  determined  in  the  following  way, 
but  at  the  same  time  the  precautions  described  in  other  works  must  be 
observed:  100  c.  c.  of  filtered  urine  is  treated  with  5  c.  c.  of  concentrated 
hydrochloric  acid  and  boiled  for  fifteen  minutes.  While  boiling  precipitate 
with  2  c.  c.  of  a  saturated  BaCl2  solution,  and  warm  for  a  little  while  until 
the  barium  sulphate  has  completely  settled.  The  precipitate  must  then  be 
washed  with  water  and  also  with  alcohol  and  ether  (to  remove  resinous 
substances),  and  then  treated  according  to  the  usual  method. 

The  separate  determination  of  the  sulphate-sulphuric  acid  and  the 
ethereal-sulphuric  acid  may  be  accomplished,  according  to  Batjmann's 
method,  by  first  precipitating  the  sulphate-sulphuric  acid  by  BaCl2  from 
the  urine  acidified  with  acetic  acid,  then  decomposing  the  ethereal-sul- 
phuric acid  by  boiling  after  the  addition  of  hydrochloric  acid,  and  finally 
determining  the  sulphuric  acid  set  free  as  barium  sulphate.  A  still  better 
method  is  the  following,  suggested  by  Salkowski:  ' 

200  c.  c.  of  urine  is  precipitated  by  an  equal  volume  of  a  barium  solution 
which  consists  of  2  vols,  barium  hydrate  and  1  vol.  barium-chloride  solu- 
tion, both  saturated  at  the  ordinary  temperature.     Filter  through  a  dry 


1  Baumann,  Zeitschr.  f.  physiol.  Chem.,  1;  Salkowski,  Virchow's  Arch.,  79. 


POTASSIUM,    SODIUM,  AND  AMMONIA.  533 

fi  tor,  measure  off  100  c.  c.  of  the  filtrate  which  contains  only  the  ethereal- 
sulphuric  acid,  treat  with  10  c.  c.  of  hydrochloric  acid  of  a  specific  gravity 
1.12,  boil  for  fifteen  minutes,  and  then  warm  on  the  water-bath  until  the 
precipitate  has  completely  settled  and  the  supernatant  liquid  is  entirely 
clear.  Filter  and  wash  with  warm  water,  and  with  alcohol  and  ether,  and 
proceed  according  to  the  generally  prescribed  method.  The  difference 
between  the  ethereal-sulphuric  acid  found  and  the  total  quantity  of  sulphuric 
acid  as  determined  in  a  special  portion  of  urine  is  taken  to  be  the  quantity 
of  sulphate-sulphuric  acid. 

Nitrates  occur  in  small  quantities  in  human  urine  (Schonbein),  and  they 
probably  originate  from  the  drinking-water  and  the  food.  According  to  \Yi\i, 
and  Citron,1  the  quantity  of  nitrates  is  smallest  with  a  meat  diet  and  greatest 
with  vegetable  food.     The  average  amount  is  about  42.5  milligrams  per  litre. 

Potassium  and  Sodium.  The  quantity  of  these  bodies  eliminated  by  the 
urine  by  a  healthy  full-grown  person  on  a  mixed  diet  is,  according  to  Sal- 
kowski,2  3-4  grams  K20  and  5-6  grams  Na20,  with  an  average  of  about 
2-3  grams  K20  and  4-6  grams  Na^O.  The  proportion  of  K  to  Na  is  ordi- 
narily as  3:5.  The  quantity  depends  above  all  upon  the  food.  In  starva- 
tion the  urine  may  become  richer  in  potassium  than  in  sodium,  which 
results  from  the  lack  of  common  salt  and  the  destruction  of  tissue  rich  in 
potassium.  The  quantity  of  potassium  may  be  relatively  increased  during 
fever,  while  after  the  crisis  the  reverse  is  the  case. 

The  quantitative  estimation  of  these  bodies  is  performed  by  the  gravi- 
metric methods  as  described  in  wTorks  on  quantitative  analysis.  In  the 
determination  of  the  total  alkalies  recently  new  methods  have  been  de- 
vised by  Pribram  and  Gregor,  and  for  the  potassium  alone  a  method  by 
Autenrieth  and  Bernheim.3  • 

Ammonia.  Some  ammonia  is  habitually  found  in  human  urine  and  in 
that  of  carnivora.  As  above  stated  (page  470),  on  the  formation  of  urea 
from  ammonia,  this  quantity  may  represent  the  small  amount  of  ammonia 
which,  because  of  the  excess  of  acids  formed  by  combustion,  as  com- 
pared with  the  fixed  alkalies,  is  united  wTith  such  acids,  and  in  this  way 
is  excluded  from  the  synthesis  to  urea.  This  view  is  confirmed  by  the 
observations  of  Coranda,  who  found  that  the  elimination  of  ammonia  was 
smaller  on  a  vegetable  diet  and  larger  on  a  rich  meat  diet' than  on  a  mixed 
diet.  On  a  mixed  diet  the  average  amount  of  ammonia  eliminated  by 
the  urine  is  about  0.7  gram  NH3  per  day  (Neubauer)  and  4.6-5.6  per  cent 
of  the  total  nitrogen  of  the  urine  according  to  Camerer,  Jr. 

As  above  stated,  all  the  ammonia  of  the  urine  is  not  represented  by 
the  residue  which  has  eluded  synthesis  into  urea  by  neutralization  with 

Schonbein,  Journ.  f.  prakt.  Chem.,  92 j  Weyl,  Virchow's  Arch.,  96,  with  Citron, 
ibid.,  101. 

'Pribram  and  Gregor,  Zeitschr.  f.  analyt.  Chem.,  38;  Autenrieth  and  Bernheim, 
Zeitschr.  f.  physiol.  Chem.,  37. 


534  URINE. 

acids,  because,  as  shown  by  Stadelmann  and  Beckmann,1  ammonia  is 
eliminated  by  the  urine  even  during  the  continuous  administration  of  fixed 
alkalies. 

Ammonia  exists  on  an  average  of  about  0.96  milligram  in  100  c.  c.  of 
human  blood,  and  in  different  amounts  in  all  the  tissues  thus  far  investi- 
gated.2 According  to  Nencki  and  Zaleski  3  it  is  abundantly  formed 
in  the  cells  of  the  digestive  glands,  the  stomach,  the  pancreas,  and  the 
intestinal  mucosa  (of  dogs)  at  the  time  when  proteid  foods  are  being  digested 
and  transported  to  the  liver.  As  the  ammonia  introduced  into  the  liver  is 
transformed  into  urea  (see  above),  we  can  therefore  expect  that  in  certain 
diseases  of  the  liver  an  increased  elimination  of  ammonia  and  a  decreased 
excretion  of  urea  will  occur.  In  how  far  this  is  true  has  already  been 
stated  (page  473),  and  we  refer  to  the  researches  of  the  various  authors 
there  cited. 

In  man  and  carnivora  the  elimination  of  ammonia  is  increased  by  the 
introduction  of  mineral  acids;  and,  as  shown  by  Jolin,  organic  acids  such 
as  benzoic  acid,  which  are  not  destroyed  in  the  body,  act  in  a  similar 
manner.  The  ammonia  set  free  in  the  proteid  destruction  is  in  part  used 
in  the  neutralization  of  the  acids  introduced,  and  in  this  way  a  destructive 
removal  of  fixed  alkalies  is  prevented.  Herbivora,  on  the  contrary,  lack 
this  property  or  have  it  only  to  a  slight  extent  (Winterberg  4).  In  them 
the  acids  introduced  are  neutralized  by  fixed  alkalies;  hence  the  introduc- 
tion of  mineral  acids  soon  causes  a  destructive  action  on  this  account. 

Acids  formed  in  the  destruction  of  proteids  in  the  body  act  on  the  elim- 
ination of  ammonia  like  those  introduced  from  without.  For  this  reason 
the  quantity  of  ammonia  in  human  and  carnivora  urine  is  increased  under 
such  conditions  and  in  such  diseases  where  an  increased  formation  of  acid 
takes  place  because  of  an  increased  metabolism  of  proteids.  This  is  the 
case  with  a  lack  of  oxygen  in  fevers  and  diabetes.  In  the  last-mentioned 
disease  organic  acids,  /?-oxybutyric  acid  and  acetoacetic  acid,  are  produced, 
which  pass  into  the  urine' combined  with  ammonia.5 

The  detection  and  quantitative  estimation  of  ammonia  used  to  be  performed 
according  to  the  method  suggested  by  Sch  osing.     The  principle  of  this  method 

1  Coranda,  Arch.  f.  exp.  Path.  u.  Pharm.,  12;  Stadelmann  (and  Beckmann),  "Ein- 
fluss  der  Alkalien  auf  den  Stoffwechsel, "  etc.  Stuttgart,  1890;  Camerer,  Zeitschr.  f. 
Biologie,  43. 

2  See  Salaskin,  Zeitschr.  f.  physiol.  Chem.,  25,  449,  and  foot-note  4,  page  202,  and 
foot-note  1 ,  page  203. 

3  Arch,  des  science  biol.  de  St.  Petersbourg,  4,  and  Salaskin,  1.  c.  See  also  Nencki 
and  Zaleski,  Arch.  f.  exp.  Path.  u.  Pharm.,  37,  and  foot-note  2,  page  348. 

'Jolin,  Skand.  Arch.  f.  Physiol.,  1;  Winterberg,  Zeitschr.  f.  physiol.  Chem.,  25. 
In  regard  to  the  behavior  of  ammonium  salts  in  the  animal  body,  see  Rumpf  and 
Kleine,  Zeitschr.  f.  Biologie,  34,  and  the  works  cited  on  page  470. 

5  On  the  elimination  of  ammonia  in  disease,  see  the  recent  works  of  Rumpf,  Vir- 
■chow's  Arch.,  143;   Hallervorden,  ibid. 


CALCIUM.      MAGNESIUM.  535 

is  that  the  ammonia  from  a  measured  amount  of  urine  is  set  free  by  lime-water 
in  a  closed  vessel  and  absorbed  by  a  measured  amount  of  N/10  sulphuric  acid. 
After  the  absorption  of  the  ammonia  the  quantity  is  determined  by  titrating  the 
remaining  free  sulphuric  acid  with  a  N/10  caustic  alkali  solution."  This  method 
gives  low  results,  and  in  exact  work  we  must  proceed  as  suggested  by  Boiiland.1 

The  recent  methods  for  estimating  the  ammonia  are  all  based  upon  the 
distillation  of  the  ammonia,  after  the  addition  of  lime,  magnesia  or  alkali 
carbonate,  at  low  temperatures  either  by  the  aid  of  vacuum  (Nencki  and 
Zaleski,  Wubsteb,  Kiu'cer  and  Reich  and  Schittkmiixm,  Schaffi  i;) 
or  by  the  aid  of  a  current  of  air  (Fo-lix)  and  then  collecting  it  in  a  standard 
acid. 

According  to  the  methods  suggested  by  Kruger,  Reich  and  Schittkx- 
HELM  2  25  c.  c.  of  the  urine  are  placed  in  a  distillation-flask  with  about  10 
grams  of  XaCl  and  1  gram  of  Na,C03  and  this  distilled  at  43°  C.  and  a 
pressure  of  30-40  milligrams  Hg  with  the  aid  of  an  air-pump.  Alcohol 
is  added  to  prevent  foaming.  The  ammonia  is  absorbed  in  N/10  acid 
contained  in  a  Peligot's  tube,  surrounded  by  ice-water,  and  when  the 
distillation  ib  finished  the  acid  is  retitrated,  making  use  of  rosolic  acid  as 
the  indicator.  In  regard  to  details,  see  the  original  publications. 
Schaffer's  method  is  practically  the  same. 

Calcium  and  magnesium  occur  in  the  urine  for  the  most  part  as  phos- 
phates. The  quantity  of  earthy  phosphates  eliminated  daily  is  somewhat 
more  than  1  gram,  and  of  this  amount  |  is  magnesium  and  J  calcium  phos- 
phate. In  acid  urines  the  mono-  as  well  as  the  di-hydrogen  earthy  phos- 
phates are  found,  and  the  solubility  of  the  first,  among  which  the  calcium 
salt  CaHP04  is  especially  insoluble,  is  particularly  augmented  by  the 
presence  in  the  urine  of  di-hydrogen  alkali  phosphates  and  sodium  chloride 
(Ott  3).  The  quantity  of  alkaline  earths  in  the  urine  depends  on  the 
composition  of  the  food.  The  lime  salts  absorbed  are  in  great  part  ex- 
creted again  into  the  intestine,  and  the  quantity  of  lime  salts  in  the  urine  is 
therefore  no  measure  of  the  absorption  of  the  same.  The  introduction  of 
readily  soluble  lime  salts  or  the  addition  of  hydrochloric  acid  to  the  food 
may  therefore  cause  an  increase  in  the  quantity  of  lime  in  the  urine,  while 
the  reverse  takes  place  on  adding  alkali  phosphate  to  the  food.  Nothing 
is  known  with  positiveness  in  regard  to  the  constant  and  regular  change  in 
the  elimination  of  calcium  and  magnesium  salts  in  disease,  and  in  these 
conditions  the  excretion  is  chiefly  dependent  upon  the  diet,  the  formation 
and  the  introduction  of  acid. 

The  quantity  of  calcium  and  magnesium  is  determined  according  to  the 
ordinary  well-known  methods. 

Iron  occurs  in  the  urine  only  in  small  quantities,  and,  as  it  seems  from  the 
investigations  of  Kunkel,  Giacosa,  Robert  and  his  pupils,  it  does  not  exist 

1  Pfliiger's  Arch.,  43,  32. 

3  Zeitschr.  f.  physiol.  Chem.,  39;  SchafTer,  Amer.  Journ.  of  Physiol.,  8,  which  con- 
tains the  literature. 

3  Zeitschr.  f.  physiol.  Chem.,  10. 


536  URINE. 

as  a  salt,  but  as  an  organic  combination — in  part  as  pigment  or  chromogen.  The 
statements  in  regard  to  the  iron  present  seem  to  show  that  the  quantity  is  very- 
variable,  from  1  to  11  milligrams  per  liter  of  urine  (Magnier,  Gottlieb,  Robert 
and  his  pupils) .  Jolles  found  as  an  average  for  twelve  persons  8  milligrams  of  iron 
in  twenty-four  hours,  while  Hoffmann,  Neumann  and  Mayer  x  find  lower  re- 
sults, an  average  of  1.02  and  0.983  milligrams.  The  quantity  of  silicic  acid  is 
ordinarily  stated  to  amount  to  about  0.03  p.  m.  Traces  of  hydrogen  peroxide  also 
occur  in  the  urine. 

The  gases  of  the  urine  are  carbon  dioxide,  nitrogen,  and  traces  of  oxy- 
gen. The  quantity  of  nitrogen  is  not  quite  1  vol.  per  cent.  The  carbon 
dioxide  varies  considerably.  In  acid  urines  it  is  hardly  one  half  as  great 
as  in  neutral  or  alkaline  urines. 

IV.  The  Quantity  and  Quantitative  Composition  of  Urine. 

The  quantity  and  composition  of  urine  is  liable  to  great  variation. 
The  circumstances  which  under  physiological  conditions  exercise  a  great 
influence  are  the  following:  the  blood-pressure,  and  the  rapidity  of  the 
blood-current  in  the  glomeruli;  the  quantity  of  urinary  constituents,, 
especially  water  in  the  blood;  and,  lastly,  the  condition  of  the  secretory 
glandular  elements.  Above  all,  the  quantity  and  concentration  of  the 
urine  depend  on  the  quantity  of  water  which  is  introduced  into  the  blood  or 
which  leaves  the  body  in  other  ways.  The  excretion  of  urine  is  increased  by 
drinking  freely  or  by  reducing  the  quantity  of  water  otherwise  removed; 
and  it  is  decreased  by  a  diminished  ingestion  of  water  or  by  a  greater 
loss  of  water  in  other  ways.  Ordinarily  in  man  just  as  much  water  is 
eliminated  by  the  kidneys  as  by  the  skin,  lungs,  and  intestine  together. 
At  lower  temperatures  and  in  moist  air,  since  under  these  conditions  the 
elimination  of  water  by  the  skin  is  diminished,  the  excretion  of  urine  may 
be  considerably  increased.  Diminished  introduction  of  water  or  increased 
elimination  of  water  by  other  ways — as  in  violent  diarrhoea  or  vomiting,  or 
in  profuse  perspiration — greatly  diminishes  the  amount  of  urine  excreted. 
For  example,  the  urine  may  sink  as  low  as  500-400  c.  c.  per  day  in  intense 
summer  heat,  while  after  copious  draughts  of  water  the  elimination  of 
3000  c.  c.  of  urine  has  been  observed  during  the  same  time.  The  quantity 
of  urine  voided  in  the  course  of  twenty-four  hours  varies  considerably 
from  day  to  day,  the  average  being  ordinarily  calculated  as  1500  c.  c.  for 
healthy  adult  men  and  1200  c.  c.  for  women.  The  minimum  elimination 
occurs  during  the  early  morning,  between  2  and  4  o'clock;  the  maximum, 
in  the  first  hours  after  waking  and  from  1-2  hours  after  a  meal. 

The  quantity  of  solids  excreted  per  day  is  nearly  constant,  even  though 


1  Kunkel  cited  from  Maly's  Jahresber.,  11;  Giacosa,  ibid.,  16;  Robert,  Arbeiten 
des  Pharm.  Instit.  zu  Dorpat,  7;  Magnier,  Ber.  d.  deutsch.  chem.  Gesellsch.,  7;  Gott- 
lieb Arch.  f.  exp.  Path.  u.  Pharm.,  20;  Jolles,  Zeitschr.  f.  anal.  Chem.,  36;  Hoffmann,. 
Zeitschr.  f.  analyt.  Chem.,  40;  Neumann  and  Mayer,  Zeitschr.  f.  physiol.  Chem.,  37. 


QUANTITY  AND  SOLIDS.  537 

the  quantity  of  urine  may  vary,  and  it  is  quite  constant  when  the  manner 
of  living  is  regular.    Therefore  the  percentage  of  solids  in  the  urine  is 

naturally  in  inverse  proportion  to  the  quantity  of  urine.  The  average 
amount  of  solid-  per  twenty-four  hours  is  calculated  as  60  grams.  The 
quantity  may  be  calculated  with  approximate  accuracy  by  means  of  the 
specific  gravity  if  the  second  and  third  decimals  of  this  factor  be  mul- 
tiplied by  Haseb's  coefficient,  2.33.  The  product  gives  the  amount 
of  solids  in  1000  c.  c.  of  urine,  and  if  the  quantity  of  urine  eliminated  in 
twenty-four  hours  be  measured,  the  quantity  of  solids  in  twenty-four  hours 
may  he  easily  calculated.  For  example,  1050  c.  c.  of  urine  of  a  specific 
gravity  1.021  was  eliminated  in  twenty-four  hours;  therefore  the  quantity 

is  0  v  10^0 
of    solids    excreted    was    21X2.33  =  48.9    and  *         =51.35    grams. 

Long  l  has  made  a  new  determination  of  the  coefficient  for  a  specific  gravity 
taken  at  25°  C.  and  finds  that  it  is  equal  to  2.6,  which  corresponds  nearly 
to  Haser's  coefficient  at  15°  C. 

Those  bodies  which,  under  physiological  conditions,  affect  the  density 
of  the  urine  are  common  salt  and  urea.  The  specific  gravity  of  the  first  is 
2.15  and  the  last  only  1.32;  so  it  is  easy  to  understand,  when  the  relative 
proportion  of  these  two  bodies  essentially  deviates  from  the  normal,  why 
the  above  calculation  from  the  specific  gravity  is  not  exact.  The  same  is 
true  when  a  urine  poor  in  normal  constituents  contains  large  amounts  of 
foreign  bodies,  such  as  albumin  or  sugar. 

A-  above  stated,  the  percentage  of  solids  in  the  urine  generally  decreases 
with  a  greater  elimination,  and  a  very  considerable  excretion  of  urine 
(polyuria)  has  therefore,  as  a  rule,  a  lower  specific  gravity.  An  important 
exception  to  this  rule  is  observed  in  urine  containing  sugar  (diabetes  meUi- 
tus),  in  which  there  is  a  copious  excretion  with  a  very  high  specific  gravity 
due  to  the  sugar.  In  cases  where  very  little  urine  is  excreted  (digvria), 
v.'s.,  during  profuse  perspiration,  in  diarrhoea,  and  in  fevers,  the  specific 
gravity  of  the  urine  is  as  a  rule  very  high;  the  percentage  of  solids  is  also 
high  and  the  urine  has  a  dark  color.  Sometimes,  as  for  example  in  certain 
cases  of  albuminuria,  the  urine  may  have  a  low  specific  gravity  notwith- 
standing the  oliguria,  and  be  poor  in  solids  and  light  in  color. 

In  certain  cases  it  is  interesting  to  know  the  relationship  between  the 
carbon  and  the  nitrogen,  or  the  quotient  C/N.  This  factor  may  vary 
between  0.7-1;  as  a  rule,  it  amounts  on  an  average  to  0.S7,  but  changes 
according  to  the  nature  of  the  food  and  is  higher  after  a  diet  rich  in  carbo- 
hydrates than  after  food  rich  in  fat  (Scholz,  Bouchard,  Pregl,  Ta\< 

It  is  difficult  to  give  a  tabular  view  of  the  composition  of  urine 

1  Journ.  Amer.  Chem.  Soc,  2."). 

'Pregl,  Pfliiger's  Arch.,  7.">,  which  contains  the  older  literature.  Tand,  Arch.  f. 
(Anat.  u.)  Physiol.,  1899,  Suppl. 


538  URINE. 

account  of  its  variation.  For  certain  purposes  the  following  table  may  be 
of  some  value,  but  it  must  not  be  overlooked  that  the  results  are  not  given 
for  1000  parts  of  urine,  but  only  approximate  figures  for  the  quantities  of 
the  most  important  constituents  which  are  eliminated  during  the  course  of 
twenty-four  hours  in  a  volume  of  1500  c.  c.  of  urine. 

Daily  quantity  of  solids=  60  grms. 
Organic  constituents=  35  grms.  Inorganic  constituents=  25  grms. 

Urea 30.0 grms.        Sodium  chloride  (NaCl).  .  .  .  15.0 grms. 

Uric  acid 0.7    "            Sulphuric  acid  (H2S04) 2.5 

Creatinine 1.0    "             Phosphoric  acid  (P203) 2.5 

Hippuric  acid 0.7    "            Potassium  (K20) 3.3 

Remaining  organic  bodies  . .     2.6    "            Ammonia  (NH3) 0.7 

Magnesia  (MgO) 0.5 

Lime  (CaO) '.  0.3 

Remaining  inorganic  bodies .  0.2 

Urine  contains  on  an  average  40  p.  m.  solids.  The  quantity  of  urea  is 
about  20  p.  m.  and  common  salt  about  10  p.  m. 

The  physico-chemical  methods  are  being  used  in  urinary  analysis  even 
to  a  greater  extent  than  in  the  analysis  of  other  animal  fluids.  A  great 
number  of  cryoscopic  determinations  but  fewer  conductivity  determinations 
have  been  made.  A  constant  relationship  between  the  values  found  by 
physico-chemical  methods  and  the  analytical  methods  has  been  sought, 
for  example,  between  the  freezing-point  depression  and  the  specific  gravity 
or  the  common  salt  and  others;  or  attempts  have  been  made  to  find  certain 
regularities  in  the  composition  of  the  urine  based  upon  the  results  of  various 
methods,  and  in  this  way  to  obtain  an  explanation  as  to  the  mechanism  of 
the  excretion  of  urine  in  order  to  apply  them  for  diagnostic  purposes.  The 
results  obtained  are,  as  is  to  be  expected,  so  variable  and  dependent  upon 
so  many  conditions  which  cannot  be  controlled,  that  certain  conclusions 
must  be  drawn  with  the  greatest  caution.  In  regard  to  the  value  and  use- 
fulness of  the  various  constants  and  relations,  which  are  based  upon 
theoretical  considerations,  the  views  are  unfortunately  still  too  divergent. 

V.  Casual  Urinary  Constituents. 

The  casual  appearance  in  the  urine  of  medicinal  agents  or  of  urinary  con- 
stituents resulting  from  the  introduction  of  foreign  substances  into  the 
organism  is  of  practical  importance,  because  such  compounds  may  inter- 
fere in  certain  urinary  investigations;  they  also  afford  a  good  means  of 
determining  whether  certain  substances  have  been  introduced  into  the 
organism  or  not.  From  this  point  of  view  a  few  of  these  bodies  will  be 
spoken  of  in  a  following  section  (on  the  pathological  urinary  constituents) . 
The  presence  of  these  foreign  bodies  in  the  urine  is  of  special  interest  in 
those  cases  in  which  they  serve  to  elucidate  the  chemical  transformations 
which    certain    substances    undergo  within   the   organism.     As   inorganic 


CASUAL  CONSTITUENTS.  5;j<) 

Bubstancea  generally  leave  the  body  unchanged,1  they  are  of  very  little 
interest  from  this  standpoint;  but  the  changes  which  certain  organic  Bub- 
stances  undergo  when  introduced  into  the  animal  body  may  thus  be  studied 
in  so  far  as  these  transformations  are  shown  by  the  urine. 

The  bodies  belonging  to  the  fatty  series  undergo,  though  not  without 
exceptions,  a  combustion  leading  towards  the  final  products  of  metab- 
olism; still,  often  a  greater  or  smaller  part  of  the  bodies  in  question  escape 
oxidation  and  appear  unchanged  in  the  urine.  A  part  of  the  acids  belong- 
ing to  this  series  which  are  otherwise  burnt  into  water  and  carbonates  and 
render  the  urine  neutral  or  alkaline  may  act  in  this  manner.  The  volatile 
fatty  acids  poor  in  carbon  are  less  easily  oxidized  than  those  rich  in  carbon, 
and  they  therefore  pass  unchanged  into  the  urine  in  large  amounts.  This 
is  especially  true  of  formic  and  acetic  acids  (Schotten,  Grehant  and 
Quixquaud2).  The  statements  in  regard  to  oxalic  acid  are  contradictory. 
In  birds,  according  to  Gaglio  and  Giuntt,  it  is  not  oxidized.  In  mammals 
it  is  in  great  part  oxidized,  according  to  Giunti,  while  Gaglio  and  Pohl 
claim  that  it  is  not  destroyed.  Marfori  and  Giunti  claim  that  in  human 
beings  oxalic  acid  is  in  great  part  oxidized,  although  the  recent  investiga- 
tions of  Salkowski,  Pierallini,  Stradomsky,  Klemperer  and  Trit- 
schler  3  seem  to  show  that  the  acid  is  only  in  part  destroyed  in  the  animal 
body.  In  order  to  exactly  determine  that  portion  of  the  ingested  oxalic 
acid  which  is  absorbed  and  excreted  by  the  urine  or  burnt  in  the  body,  t 
must  necessarily  be  known  whether  or  not  a  portion  of  the  acid  is  destroyed 
in  the  intestine  and  is  therefore  not  absorbed.  Tartaric  acids  act  differ- 
ently, according  to  Brion;  4  namely,  in  dogs  the  kevo-tartaric  acid  is  nearly 
entirely  consumed,  while  a  little  more  than  70  per  cent  of  dextro-tartaric 
acid  is  burnt.  Racemic  acid  is  oxidized  to  a  still  less  extent  in  the  animal 
body.  Succinic  and  malic  acids  are  completely  combustible,  according  to 
Pohl.5  Examples  of  the  different  behavior  of  stereoisomeric  substances 
have  already  been  given  on  page  88. 

The  acid  amides  appear  not  to  be  altered  in  the  body  (Schultzex  and 
Nencki  6).  A  small  part  of  the  amino  acids  seems  to  be  eliminated  un- 
changed, but  otherwise,  as  stated   above  (pa^e  469)  for    leucin,  glycocoU, 

1  In  regard  to  the  behavior  of  certain  of  these  bodies,  see  HefTter,  Die  Ausscheidung 
korperfremden  Substanzen  in  Ham,  Ergebnisse  d.  Physiol.,  2,  Abt.  I. 

2  Schotten,  Zeitschr.  f.  physiol.  Chem.,  7;  G  reliant  and  Quinquaud,  Compt.  rend., 
104. 

3  Gaglio,  Arch.  f.  exp.  Path.  u.  Pharm.,  22;  Giunti,  Chem.  Centralbl.,  1S97,  2; 
.Marfori,  Maly's  Jahresber.,  20  and  27;  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Sal- 
kowski, Berl.  klin.  Wochenschr.,  1900;  Pierallini,  Virchow's  Arch.,  160;  Stradomsky, 
ibid.,  103;    Klemperer  and  Tritschler,  Zeitschr.  f.  klin.  Med.,  44. 

4  Zeitschr.  f.  physiol.  Chem.,  2."j. 

5  Pohl,  Arch,  f  exp.  Path.  u.  Pharm.,  37,  which  also  contains  the  statements  on 
the  intermediary  products  formed  in  the  oxidation  of  the  fatty  bodies. 

8  Zeitschr.  f.  Biologie,  8. 


540  URINE. 

and  aspartic  acid,  they  are  decomposed  within  the  body,  and  may  there- 
fore cause  an  increased  excretion  of  urea.  Sarcosin  (methylglycocoll) , 
NH(CH3).CH2.COOH,  also  perhaps  passes  in  small  part  into  the  corre- 
sponding uramino  acid,  methylhydantoic  acid,  NH2.CO.N(CH3).CH2.COOH 
(Schultzen  1).  Likewise  taurin,  aminoethylsulphonic  acid,  which  acts 
somewhat  differently  in  different  animals  (Salkowski  2),  passes  in  human 
beings,  at  least  in  part,  into  the  corresponding  uramino  acid,  tauro- 
carbamic  acid,  NH2.CO.NH.C2H4.S02.OH.  A  part  of  the  taurin  also 
appears  as  such  in  the  urine.  In  rabbits,  when  taurin  is  introduced  into 
the  stomach  nearly  all  its  sulphur  appears  in  the  urine  as  sulphuric  and 
hyposulphurous  acids.  After  subcutaneous  injection  the  taurin  appears 
again  in  great  part  unchanged  in  the  urine. 

The  nitriles,  including  hydrocyanic  acid,  pass,  according  to  Lang,  into 
sulphocyanide  combinations,  and  this  sulphocyanide  apparently  originates 
from  the  non-oxidized  sulphur  of  the  proteids,  which  is  readily  split  off. 
Pascheles'  observations  indicate  that,  in  an  alkaline  reaction  and  at  the 
temperature  of  the  body,  this  sulphur  can  convert  the  alkali-cyanides 
readily  into  sulphocyanides.  The  alkali  sulphocyanides  when  ingested 
are  nearly  quantitatively  eliminated  in  the  urine,  according  to  Pollak.3 

By  substitution  with  halogens,  bodies  otherwise  readily  oxidizable  are 
converted  into  difficultly  oxidizable  ones.  While  the  aldehydes  are  readily 
and  completely  burnt  like  the  primary  and  secondary  alcohols  of  the  fatty 
series,  the  halogen  substituted  aldehydes  and  alcohols  are,  on  the  contrary, 
difficultly  oxidizable.  The  halogen  substitution  products  of  methane 
(chloroform,  iodoform,  and  bromoform)  are  at  least  in  part  burnt,  and  the 
corresponding  alkali  combination  of  the  halogen  passes  into  the  urine.4 

By  coupling  with  sulphuric  acid,  the  alcohols  which  are  otherwise  readily 
oxidizable  may  be  guarded  against  combustion,  and  consequently  the  alkali 
salt  of  ethylsulphuric  acid  is  not  burnt  in  the  body  (Salkowski  5). 

The  organic  combinations  containing  sulphur  act  somewhat  differently. 
W.  Smith  states  that  the  sulphur  of  the  thio  acids  like  thioglycolic  acid, 
CH2.SH.COOH,  is  in  part  oxidized  to  sulphuric  acid,  and  according  to 
Goldmanx  the  same  result  occurs  with  aminothiolactic  acid  (cystein) 
and  the  sulphur  of  the  thio  alcohols  (ethyl  mercaptans).  On  the 
contrary,  ethylsulphide,  sulphonic  and  sulpho  acids  in  general  (Salkowski, 

1  Ber.  d.  deutsch.  chem.  Gesellsch.,  5.  See  also  Baumann  and  v.  Mering,  ibid.,  8, 
584,  and  E.  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  4,  107. 

2  Ber.  d.  deutsch.  chem.  Gesellsch.,  6,  and  Virchow's  Arch.,  58. 

3  Lang,  Arch.  f.  exp.  Path.  u.  Pharm.,  34;  Pascheles,  ibid.;  Pollak,  Hofmeister's 
Beitriige,  2. 

4  See  Harnack  and  Griindler,  Berlin,  klin.  Wochenschr. ,  1883;  Zeller,  Zeitschr.  f. 
physiol.  Chem.,  8;  Kast,  ibid.,  11;  Binz,  Arch.  f.  exp.  Path.  u.  Pharm.,  28;  Zeehuisen, 
Maly's  Jahresber.,  23. 

6  Pfluger's  Arch.,  4. 


CA8UAL  CONSTITUENTS.  541 

Smith1)  are  not  changed  into  sulphuric  acid.  Oxyethylsulphonic  acid, 
HO.G3H4.SO3.OH,  which  is  in  part  oxidized  to  sulphuric  acid,  is  an  excep- 
tion (Salkowski). 

Conjugation  ivith  glucuronic  acid  occurs,  according  to  the  investigations 
of  Sundvik  and  especially  of  0.  Neubauer,  in  many  substituted  as  well 
as  non-substituted  alcohols,  aldehydes,  and  ketones.  Chloral  hydrate, 
('.,( 'l.,(  >l I  ll,<),  passes,  after  it  has  been  converted  into  trichlorethyl- 
alcohol  by  a  reduction,  into  a  lsevogyrate  reducing  acid,  urocMoralic  add 
or  trichlorethyl-glucuronic  acid,  C2C13H2.C6H907  (Musculus  and  v.  Mering). 
Of  the  primary  alcohols  investigated  by  Neubauer  2  (upon  rabbits  and 
dogs)  methyl  alcohol  gave  no  conjugated  glucuronic  acid  and  ethyl  alcohol 
only  a  small  amount.  Isobutyl  alcohol  and  active  amyl  alcohol  yielded 
relatively  large  quantities.  Secondary  alcohols  produced  conjugated  glucu- 
ronic acids,  and  indeed  to  a  greater  extent  than  the  primary  alcohols,  espe- 
cially in  rabbits.  The  ketones  are  reduced  in  part  into  secondary  alcohols 
and  are  partly  excreted  as  the  conjugated  acid.  This  could  be  shown 
for  acetone  with  rabbits  but  not  with  dogs. 

The  homo-  and  heterocyclic  compounds  pass,  as  far  as  is  known,  into 
the  urine  as  such,  or,  after  a  previous  partial  oxidation  or  synthesis  with 
other  bodies,  they  appear  as  so-called  aromatic  compounds.  That  the 
benzene  ring  is  destroyed  in  the  body  in  certain  cases  is  very  probable. 

The  fact  that  benzene  may  be  oxidized  outside  of  the  body  into  carbon 
dioxide,  oxalic  acid,  and  volatile  fatty  acids  has  been  known  for  a  long 
time;  and  as  in  these  cases  a  rupture  of  the  benzene  ring  must  take  place, 
so  also,  it  must  be  admitted,  when  aromatic  substances  undergo  a  com- 
bustion in  the  animal  body,  a  splitting  of  the  benzene  nucleus  with  the 
formation  of  fatty  bodies  must  be  the  result.  If  this  does  not  occur, 
then  the  benzene  nucleus  is  eliminated  with  the  urine  as  an  aromatic  com- 
bination of  one  kind  or  another.  As  the  benzene  nucleus  can  protect  from 
destruction  a  substance  belonging  to  the  fatty  series  when  conjugated  with 
it,  which  is  the  case  with  the  glycocoll  of  hippuric  acid,  it  seems  also  that 
the  aromatic  nucleus  itself  may  be  protected  from  oxidation  in  the  organ- 
ism by  syntheses  with  other  bodies.  The  aromatic  ethereal-sulphuric  acids 
are  examples  of  this  kind. 

The  difficulty  in  deciding  whether  the  benzene  ring  itself  is  destroyed  in 
the  body  lies  in  the  fact  that  we  do  not  know  all  the  different  aromatic 
transformation  products  which  may  be  produced  by  the  introduction  of 

1  Smith,  Pfluger's  Arch.,  53,  00,  57,  and  Zeitschr.  f.  phvsiol.  Chem.,  17;  Salkowski, 
Virchow's  Arch.,  66;  Pfluger's  Arch.,  39;  Goldmann,  Zeitschr.  f.  physiol.  Chem.,  9; 
also  Baumann  and  Kast,  ibid.,  14. 

2 Sundvik,  Maly's  Jahresber.,  16;  Musculus  and  v.  Mering,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  8;  also  v.  Mering,  ibid.,  15,  Zeitschr.  f.  physiol.  Chem  ,  6;  Kiilz,  Pfluger's 
Arch.,  2S  and  33;   O.  Neubauer,  Arch.  f.  exp.  Path.  u.  Pharm.,  46. 


542  URINE. 

any  such  substance  into  the  organism,  and  which  must  be  sought  for  in 
the  urine.  On  this  account  it  is  also  impossible  to  learn  by  exact  quanti- 
tative determinations  whether  or  not  an  aromatic  substance  ingested 
or  absorbed  appears  again  unchanged  in  the  urine.  Certain  observa- 
tions render  it  probable  that  the  benzene  ring,  as  above  mentioned,  is  at 
least  in  certain  cases  destroyed  in  the  body.  Schotten,  Baumann,  and 
others  have  found  that  certain  amino  acid  ■,  :  uch  as  phenylamino-propionic 
acid  and  amino-cinnamic  acid,  and  tyrosin  when  introduced  into  the  body 
cause  no  increase  in  the  quantity  of  known  aromatic  substances  in  the 
urine;  this  makes  a  destruction  of  these  amino  acids  in  the  animal  body 
seem  probable.  Juvalta  also  made  experiments  on  dogs  with  phthalic 
acid  and  found  that  it  was  in  great  part  destroyed.  The  benzene  deriv- 
atives vary  in  behavior  according  to  the  position  of  the  substitution,  for  as 
found  by  R.  Cohn,1  among  the  di-derivates  the  ortho  compounds  are  more 
readily  destroyed  than  the  corresponding  meta  or  para  compounds. 

An  oxidation  in  the  side  chain  of  aromatic  compounds  is  often  found, 
and  may  also  occur  in  the  nucleus  itself.  As  an  example,  benzene  is  first 
oxidized  to  oxybenzene  (Schultzen  and  Naunyn),  and  this  is  then  further 
in  part  oxidized  into  dioxybenzenes  (Baumann  and  Preusse).  Naph- 
thalene appears  to  be  converted  into  oxynaphthalene,  and  probably  a  part 
also  into  dioxynaphthalene  (Lesnik  and  M.  Nencki).  The  hydrocarbon 
with  an  amino  or  imino  group  may  also  be  oxidized  by  a  substitution  of 
hydroxyl  for  hydrogen,  especially  when  the  formation  of  a  derivative  in 
the  para  position  is  possible  (Klingenberg).  For  example,  aniline, 
C6H5.NH2,  passes  into  paraminophenol,  which  latter  passes  into  the  urine  as 
its  ethereal-sulphuric  acid,  H2N.C6H4.O.S02.OH  (F.  Muller).  Acetanilid  is 
in  part  converted  into  acetyl  paraminophenol  (Jaffe  and  Hilbert,  K 
Morner)  and  carbazol  into  oxycarbazol  (Klingenberg  2). 

An  oxidation  of  the  side  chain  may  occur  by  the  hydrogen  atoms  being 
replaced  by  hydroxyl  as  in  the  oxidation  of  indol  and  skatol  into  indoxyl 
and  skatoxyl.  An  oxidation  of  the  side  chain  may  also  take  place  with  the 
formation  of  carboxyl;  thus,  for  example,  toluene,  C6H5.CH3  (Schultzen  and 
Naunyn),  ethyl-benzene,  C6H5.C2H5,  and  propylbenzene,  C6H5.C3H7  (Nencki 
and  Giacosa3),  besides  many  other  bodies,  are  oxidized  into  benzoic  acid. 

1  Schotten,  Zeitschr.  f.  physiol.  Chem.,  7  and  8;  Baumann,  ibid. ,"10,  130.  In  regard 
to  the  behavior  of  tyrosin,  see  especially  Blcndermann,  ibid.,  6;  Schotten,  ibid.,  7; 
Bass,  ibid  ,  11;  and  R.  Cohn,  ibid.,  14,  17;  Juvalta,  ibid.,  13. 

-  Schultzen  and  Naunyn,  Reichert  and  Du  Bois-Reymond's  Arch.,  1867;  Baumann 
and  Preusse,  Zeitschr.  f.  physiol.  Chem.,  3;  156.  See  also  Nencki  and  Giacosa,  ibid.,  4; 
Lesnik  and  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  24;  F.  Muller,  Deutsch.  med. 
Wochenschr.,  1887;  Jaffd  and  Hilbert,  Zeitschr.  f.  physiol.  Chem.,  12;  Morner,  ibid., 
13;  Klingenberg,  "Studien  uber  die  Oxydation  aromatischer  Subsfranzen,"  etc. 
Inaug.-Diss.  Rostock,  1891.  In  regard  to  formanilid,  which  acts  essentially  as 
acetanilid,  see  Kleine,  Zeitschr.  f.  physiol    Chem.,  22. 

3  Ibid.,  4. 


CASUAL  CONSTITUENTS.  543 

Cymenc  is  oxidized  to  cumic  acid,  xylene  to  toluic  acid,  methylpyridine  to 
pyridine-carbonic  acid  in  the  same  way.  If  the  Bide  chain  has  several 
members,  the  behavior  is  somewhat  different.  Phenylacetic  acid,  C6H5. 
CH2.COOH,  in  which  only  one  carbon  atom  exists  between  the  benzene 
nucleus  and  the  carboxyl,  is  not  oxidized  but  is  eliminated  after  conjuga- 
tion with  glycocoll  as  phenaccturic  acid  (SALKOWSKI  l).  Phenylpropionic 
acid,  C6H5.CH2.CH2.COOH,  with  two  carbon  atoms  between  the  benzene 
nucleus  and  the  carboxyl,  is,  on  the  contrary,  oxidized  into  benzoic  acid.2 
Aromatic  amino  acids  with  three  carbon  atoms  in  the  side  chain,  and 
in  which  the  NH2  group  is  bound  to  the  middle  one,  as  in  tyrosin, 
o-oxyphenylaminopropionic  acid,  C6H4(OH).CHrCH(NH2).COOH,  and 
a-phcniiUnninopropionic  acid,  C6H5.CH2.CH(NH2).COOH,  seem  to  be  in  great 
part  burnt  within  the  body  (see  above).  Phenylaminoacetic  acid,  which 
has  only  two  carbon  atoms  in  the  side  chain,  C0H5.CH(NH2)COOH, 
acts  differently,  passing  into  mandelic  acid,  phenylglycolic  acid, 
C6H5.CH(OH).COOH  (Schotten  3). 

If  several  side  chains  are  present  in  the  benzene  nucleus,  then  only  one 
is  always  oxidized  into  carboxyl.  Thus  xylene,  C6H4(CH3)2,  is  oxidized 
into  toluic  acid,  C0H4(CH5)COOH  (Schultzen  and  Naunyn),  mesitylene, 
C6H3(CH3)3,  into  mesitylenic  acid,  C6H3(CH3)2.COOH  (L.  Nencki),  and 
cymene  into  cumic  acid  (M.  Nencki  and  Ziegler  4). 

Syntheses  of  aromatic  substances  with  other  atomic  groups  occur  fre- 
quently. To  these  syntheses  belongs,  in  the  first  place,  the  conjugation  of 
benzoic  acid  with  glycocoll  to  form  hippuric  acid,  first  discovered  by  Woh- 
ler.  All  the  numerous  aromatic  substances  which  are  converted  into 
benzoic  acid  in  the  body  are  voided  partly  as  hippuric  acid.  This  state- 
ment is  not  true  for  all  species  of  animals.  According  to  the  observations 
of  Jaffe,5  benzoic  acid  does  not  pass  into  hippuric  acid  in  birds,  but  into 
another  nitrogenous  acid,  ornithuric  acid,  C19H20N2O4.  This  acid  yields  as 
splitting  products,  besides  benzoic  acid,  ornithin,  a  body  which  has  been 
spoken  of  on  page  78.  Not  only  are  the  oxybenzoic  acids  and  the  sub- 
stituted benzoic  acids  conjugated  with  glycocoll,  forming  corresponding 
hippuric  acids,  but  also  the  above-mentioned  acids,  toluic,  mesitylenic, 
cumic.  and  phenylacetic  acids.  These  acids  are  voided  as  toluric,  mesitul- 
enuric,  cuminuric,  and  phenaceturic  acids. 

It  must  be  remarked  in  regard  to  the  oxybenzoic  acids  that  a  con- 
jugation with  glycocoll  has  only  been  shown  with    salicylic  and  p-oxy- 


1  Zeitschr.  f.  physiol.  Chem.,  7  and  9. 

2  See  E.  and  H.  Salkowski,  Ber.  d.  deutsch.  chem.  Gesellseh.,  12. 

3  Zeitschr.  f.  physiol.  Chem.,  8. 

4L.  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  1;  Xencki  and  Ziegler,  Ber.  d.  deutsch. 
chem.  Gesellseh.,  o.     See  also  O.  Jacobsen,  ibid.,  12. 
*Ibid.,  10  and  11. 


544  URINE. 

benzoic  acid  (Bertagnini,  Batjmann,  Herter,  and  others),  while  Bau- 
manx  and  Herter  *  find  it  only  very  probable  for  m-oxybenzoic  acid. 
The  oxybenzoic  acids  are  also  in  part  eliminated  as  conjugated  sulphuric 
acids,  which  is  especially  true  for  m-oxybenzoic  acid.  The  three  amino- 
benzoic  acids,  according  to  the  experiments  of  Hildebraxdt,  on  rabbits, 
appeared  at  least  in  part  unchanged  in  the  urine.  Salkowski  found, 
as  was  later  confirmed  by  R.  Cohx,2  that  m-aminobenzoic  acid  passes  in 
part  into  uraminobenzoic  acid,  H2N.CO.HN.C6H4.COOH.  It  is  also  in  part 
eliminated  as  aminohippuric  acid. 

The  halogen  substituted  compounds  of  toluene  behave  somewhat  differ- 
ent in  various  animals  according  to  Hildebraxdt 's  experiments.  In  dogs 
they  are  converted  into  the  corresponding  substituted  hippuric  acid.  In 
rabbits  o-bromtoluene  is  completely  changed  to  hippuric  acid,  the  m-  and 
p-bromtoluene  only  partly.  The  three  chlortoluenes  are  converted  in  rab- 
bits into  the  corresponding  benzoic  acid  and  are  eliminated  as  such  and 
not  as  hippuric  acid. 

The  substituted  aldehydes  are  of  special  interest  as  substances  which 
may  undergo  conjugation  with  glycocoll.  According  to  the  investigations 
of  R.  Cohn3  on  this  subject  o-nitrobenzaldehyde  when  introduced  into  a 
rabbit  is  only  in  a  very  small  part  converted  into  nitrobenzoic  acid,  and 
the  chief  mass,  about  90  per  cent,  is  destroyed  in  the  body.  According 
to  Sieber  and  Smirxow  4  m-nitrobenzaldehyde  passes  in  dogs  into  m-nitro- 
hippuric  acid,  and  according  to  Cohn  into  urea-  ra-nitrohippurate.  In 
rabbits  the  behavior  is  quite  different.  In  this  case  not  only  does  an 
oxidation  of  the  aldehyde  into  benzoic  acid  take  place,  but  the  nitro 
group  is  also  reduced  to  an  amino  group,  and  finally  to  this  acetic  acid 
attaches  itself  with  the  expulsion  of  water,  so  that  the  final  product 
is  m-acetylaminobenzoic  acid,  CH3.CO.NH.C6H4.COOH.  This  process  is 
analogous  to  the  behavior  of  furfurol,  and  the  reduction  does  not  take 
place  in  the  intestine,  but  in  the  tissues.  The  p-nitrobenzaldehyde 
acts  in  rabbits  in  part  like  the  m-aldehyde  and  passes  in  part  into 
p-acetylaminobenzoic  acid.  Another  part  is  converted  into  p-nitro- 
benzoic  acid,  and  the  urine  contains  a  chemical  combination  of  equal 
parts  of  these  two  acids.  According  to  Sieber  and  Smirnow  p-nitro- 
benzaldehyde  yields  only  urea  p-nitrohippurate  in  dogs.  The  above- 
mentioned  pyridine-carbonic  acid,  formed  from  methylpyridine  (a-picoline) 
passes  into  the  urine  after  conjugation  with  glycocoll  as  a-pyridineuric  acid.5 

1  Zeitschr.  f.  physiol.  Chem.,  1,  where  Bertagnini's  work  is  also  cited.  See  also 
Dautzenberg,  Maly's  Jahresber.,  11,  231. 

2  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  7j  Cohn,  ibid.,  17;  Hildebrandt,  Hof- 
meister's  Beitrage,  3. 

3  Zeitschr.  f.  physiol    Chem.,  17. 

4  Monatshefte  f.  Chem.,  8. 

6  In  regard  to  the  extensive  literature  on  glycocoll  conjugations  we  refer  the  reader 


CASUAL   CONSTITUENTS.  545 

To  those  substances  which  undergo  a  conjugation  with  glycocoll  belongs 

also  jurjurol  (the  aldehyde  of  pyromucic  acid),  which,  when  introduced  into 
rabbits  and  dogs,  as  shown  by  Jaffe  and  Cohn,  is  first  oxidized  into  pyro- 
mucic acid  and  then  eliminated  as  pyromucuric  acid,  C7II7X/),  after 
conjunction  with  glycocoU.  In  birds  this  behavior  is  different,  namely, 
the  acid  is  conjugated  with  another  substance,  ornithin,  C5H12X202,  which 
is  a  diaminovalerianic  acid,  forming  pyromucinornithuric  acid.  Similar  to 
furfurol,  thiophene,  C4H4S,  corresponding  to  furfurane,  is  oxidized  to  thio- 
phenic  acid,  which,  according  to  Jaffe  and  Levy,1  is  conjugated  with  glyco- 
coU in  the  body  (rabbits)  and  eliminated  as  thiophenuric  acid,  C7II7.\S<)3. 

Furfurol  also  undergoes  conjugation  with  glycocoU  in  other  forms  in 
mammals.  Thus  Jaffe  and  Cohn  found  that  it  is  in  part  combined  with 
acetic  acid,  forming  furfuracrylic  acid,  C4H3O.CH:CH.COOH,  which  passes 
into  the  urine  coupled  with  glycocoU  as  furfuracryluric  acid. 

Another  very  important  synthesis  of  aromatic  substances  is  that  of 
the  ethereal-sulphuric  acids.  Phenols  and  chiefly  the  hydroxylated  aromatic 
hydrocarbons  and  their  derivatives  are  voided  as  ethereal-sulphuric  acids, 
according  to  Baumann,  Herter,  and  others.2 

A  conjugation  of  aromatic  acids  with  sulphuric  acid  occurs  less  often. 
The  two  above-mentioned  aromatic  acids,  p-oxyphenylacetic  and  p-oxy- 
phcnylpropionic  acid,  are  in  part  eliminated  in  this  form.  Gentisic  acid 
(hydroquinone-carbonic  acid)  also  increases,  according  to  Likhatscheff,3 
the  quantity  of  ethereal-sulphuric  acid  in  the  urine,  and  according  to 
Rost  the  same  occurs,  contrary  to  the  older  statements,  with  gallic  acid 
(trioxybenzoic  acid)   and  tannic  acid.* 

While  acetophenone  (phenylmethylketone) ,  C6H3.CO.CH3,  as  shown  by 
M.  Nencki,  is  oxidized  to  benzoic  acid  and  eliminated  as  hippuric  acid, 
the  aromatic  oxyketones  with  hydroxyl  groups,  such  as  resacetophenone, 

C6H3(OH)(OH)(CO.CH3),    paraoxypropiophenone,    C6H4(OH)(COCH2.CH3), 

12  3  4 

and  gallacetophcnone,  C6H2(OH)(OH)(OH)(CO.CH3),  pass  into  the  urine 
without  previous  oxidation  as  ethereal-sulphuric  acids  and  in  part  after 
conjugation  with  glucuronic  acid  (Nencki  and  Rekowski  5).     Euxanthon, 

to  O.  Kuhling.  Ueber  Stoffwechselprodukte  aromatischer  Korper.  Inaug.-Diss., 
Berlin,  18S7. 

1  JafT6  and  Cohn,  Ber.  d.  deutsch.  chem  Gesellsch.,  20  and  21;  with  Levy,  ibid.,  21. 

3  In  regard  to  the  literature,  see  O.  Kiihling,  1.  c. 
s  Zeitschr.  f.  physiol.  Chem.,  21. 

4  In  regard  to  the  behavior  of  gallic  and  tannic  acids  in  the  animal  body,  see  C. 
Morner,  Zeitschr.  f.  physiol.  Chem.,  16,  which  also  contains  the  older  literature;  also 
Harnack,  ibid.,  24,  and  Rost,  Arch.  f.  exp  Path.  u.  Pharm.,  38,  and  Sitzungsber  d. 
Gesellsch.  zur  Beford.  d.  ges.  Xaturwiss.  zu  Marburg,  1S98. 

s  Arch.  d.  scienc.  biol.  de  St.  Ptftersbourg,  3,  and  Ber.  d.  deutsch.  chem.  Gesellsch., 
27. 


54G  URINE. 

which  is  also  an  aromatic  oxyketone,  passes  into  the  urine  as  euxanthic  acid 
after  the  conjugation  with  glucuronic  acid  previously  mentioned. 

A  conjugation  of  other  aromatic  substances  with  glucuronic  acid,  which 
last  is  protected  from  combustion,  occurs  rather  often.  The  phenols,  as 
above  stated  (page  505),  pass  in  part  as  conjugated  glucuronic  acids  into 
the  urine.  The  same  is  true  for  the  homologues  of  the  phenols,  for  certain 
substituted  phenols,  and  for  many  aromatic  substances,  also  hydrocarbons 
after  previous  oxidation  and  hydration.  Thus  Hildebrandt  and  Fromm: 
and  Clemens  *  have  shown  that  the  cyclic  terpenes  and  camphors,  by  oxida- 
tion or  hydration,  or  in  certain  cases  by  both,  are  converted  into  hydroxyl 
derivatives,  when  the  body  in  question  is  not  previously  hydroxylized,  and 
that  these  hydroxyl  derivatives  are  eliminated  as  conjugated  glucuronic 
acids.  Conjugated  glucuronic  acids  are  detected  in  the  urine  after  the 
introduction  of  various  substances,  e.g.,  therapeutic  agents,  into  the  organ- 
ism, namely,  terpenes,  borneol,  menthol,  camphor  (camphoglucuronic  acid 
was  first  observed  by  Schmiedeberg),  naphthalene,  oil  of  turpentine,  oxy- 
quinolines,  antipyrine,  and  many  other  bodies.2  Orthonitrotoluene  in  dogs 
passes  first  into  o-nitrobenzyl  alcohol  and  then  into  a  conjugated  glucuronic 
acid,  uronitrotoluolic  acid  (Jaffe  3).  The  glucuronic  acid'  split  off  from 
this  conjugated  acid  is  laevogyrate  and  hence  not  identical,  but  only  isomeric 
with  the  ordinary  glucuronic  acid.  Indol  and  skatol  seem,  as  above  stated 
(page  509),  to  be  eliminated  in  the  urine  partly  as  conjugated  glucuronic 
acids. 

A  synthesis  in  which  compounds  containing  sulphur,  mercapturic  acid,  are 
formed  and  eliminated  after  conjugation  with  glucuronic  acid,  occurs  when 
chlorine  and  bromine  derivatives  of  benzene  are  introduced  into  the  organism 
of  dogs  (Batjmann  and  Preusse,  Jaffe).  Thus  chlorbenzene  combines 
with  cystein,  forming  chlorphenylmercapturic  acid,  CnHi2ClSN03.  The 
recent  investigations  of  Friedmann  4  show  that  the  phenylthiolactic  acid 
which  forms  the  foundation  of  the  mercapturic  acids  belongs  to  the  /3-series, 
and  in  this  way  the  direct  chemical  connection  of  this  body  with  the  pro- 
teid-cystin  (a-amino-/?-thiolactic  acid)  is  established.  Friedmann  has  also 
been  able  to  convert  cystein  into  bromphenylmercapturic  acid.. 

Pyridine,  C5H5N,  which  does  not  combine  either  with  glucuronic  acid 
or  with  sulphuric  acid  after  previous  oxidation,  shows  a  special  behavior. 

'Hildebrandt,  Arch.  f.  exp.  Path.  u.  Pharm.,  45,  46;  Zeitschr.  f.  physiol.  Chem., 
36;  with  Fromm,  ibid.,  33;  and  with  Clemens,  ibid.,  37;  Fromm  and  Clemens,  ibid.,  oi. 

2  See  O.  Kiihling,  1.  c,  which  gives  the  literature  up  to  18S7;  also  E.  Kiilz,  Zeitschr. 
f.  P.iologie,  27;  the  works  of  Hildebrandt,  Fromm  and  Clemens,  see  foot-note  1; 
Brahm,  Zeitschr.  f.  physiol.  Chem.,  28;  Fenyvessy,  ibid.,  30;  Bonanni,  Hofmeister's 
Beitrage,  1;   Lawrow,  Ber.  d.  d.  chem.  Gesellsch.,  33. 

'  Zeitschr.  f.  physiol.  Chem.,  2. 

i  Baumann  and  Preusse,  Zeitschr.  f.  physiol.  Chem.,  5;  Jaff6,  Ber.  d  deutsch. 
chem.  Gesellsch.,  12;    Friedmann,  Hofmeister's  Beitrage,  4. 


PATHOLOGICAL  CONSTITUENTS.  547 

It  takes  up  a  methyl  group  as  found  by  J  lis  and  Inter  confirmed  byConx,1 
and  forms  an  ammonium  combination,  mcthylpyr  idyl-ammonium  hydroxide} 
II<U'II,..\C,II,V 

Several  alkaloids,  such  as  quinine,  morphine,  and  strychnine,  may  pass 
into  the  urine.  After  the  ingestion  of  turpentine,  balsam  of  copaiva,  and 
resins,  these  may  appear  in  the  urine  as  resin  acids.  Differenl  kinds  of 
coloring-matters,  such  as  alizarin,  crysophanic  acid,  after  rhubarb  or  senna, 
and  the  coloring-matter  of  the  blueberry,  etc.,  may  also  pass  into  the  urine. 
After  rhubarb,  senna,  or  santonin  the  urine  assumes  a  yellow  or  greenish- 
yellow  color,  which  is  transformed  into  a  beautiful  red  by  the  addition 
of  alkali.  Phenol  produces,  as  above  mentioned,  a  dark-brown  or  dark- 
green  color  which  depends  mainly  on  the  decomposition  products  of  hydro- 
quinone  and  humin  substances.  After  naphthalene  the  urine  has  a  dark 
color,  and  several  other  medicinal  agents  produce  a  special  coloration. 
Thus  after  antipyrine  it  becomes  yellow  or  blood-red.  After  balsam  of 
copaiva  the  urine  becomes,  when  strongly  acidified  with  hydrochloric  acid, 
gradually  rose  and  purple-red.  After  naphthalene  or  naphthol  the  urine 
gives  with  concentrated  sulphuric  acid  (1  c.  c.  of  concentrated  acid  and  a 
few  drops  of  urine)  a  beautiful  emerald-green  color,  which  is  probably  due 
to  naphthol-glucuronic  acid.  Odoriferous  bodies  also  pass  into  the  urine. 
After  asparagus  the  urine  acquires  a  sickly  disagreeable  odor  which  is  prob- 
ably due  to  methylmercaptan,  according  to  M.  Nencki.2  After  turpentine 
the  urine  may  have  a  peculiar  odor  similar  to  that  of  violets. 

VI.   Pathological  Constituents  of  Urine. 

Proteid.  The  appearance  of  slight  traces  of  proteid  in  normal  urines 
has  been  repeatedly  observed  by  many  investigators,  such  as  Posxer, 
Plosz,  v.  Noordex,  Leube,  and  others.  According  to  K.  M6RNEB  3  pro- 
teid regularly  occurs  as  a  normal  urinary  constituent  to  the  extent  of  22-78 
milligrams  per  liter.  Frequently  traces  of  a  substance  similar  to  a  nucleo- 
albumin,  which  is  easily  mistaken  for  mucin,  appears  in  the  urine  and 
whose  nature  will  be  treated  of  later.  In  diseased  conditions  proteid 
occurs  in  the  urine  in  a  variety  of  cases.  The  albuminous  bodies  which 
most  often  occur  are  serglobulin  and  seralbumin.  Albumoses  (or  pep- 
tones) also  sometimes  are  present.  The  quantity  of  proteid  in  the  urine 
is  in  most  cases  less  than  5  p.  m.,  rarely  10  p.  m.,  and  only  very  rarely 
does  it  amount  to  50  p.  m.  or  over.  Cases  are  known,  however,  where  it 
was  even  more  than  80  p.  m. 

Among  the  many  reactions  proposed  for  the  detection  of  proteid  in 
urine,  the  following  are  to  be  recommended: 

1  His,  Arch.  f.  exp.  Path.  u.  Pharm.,  22;  Cohn,  Zeitschr.  f.  physiol.  Chem.,  18. 
"Arch.  f.  exp.  Path.  u.  Pharm.,  98. 
sSkand.  Arch.  f.  Physiol.,  6  (literature). 


548  URINE. 

The  Heat  Test.  Filter  the  urine  and  test  its  reaction.  An  acid  urine 
may,  as  a  rule,  be  boiled  without  further  treatment,  and  only  in  especially 
acid  urines  is  it  necessary  to  first  treat  with  a  little  alkali.  An  alkaline 
urine  is  made  neutral  or  faintly  acid  before  heating.  If  the  urine  is  poor 
in  salts,  add  TV  vol.  of  a  saturated  common-salt  solution  before  boiling;  then 
heat  to  the  boiling-point,  and  if  no  precipitation,  cloudiness,  or  opalescence 
appears,  the  urine  in  question  contains  no  coagulable  proteid,  but  it  may 
contain  albumoses  or  peptones.  If  a  precipitate  is  produced  on  boiling,  this 
may  consist  of  proteid,  or  of  earthy  phosphates,  or  of  both.  The  mono- 
hydrogen  calcium  phosphate  decomposes  on  boiling,  and  the  normal  phos- 
phate may  separate  out.  The  proper  amount  of  acid  is  now  added  to  the 
urine,  so  as  to  prevent  any  mistake  caused  by  the  presence  of  earthy  phos- 
phates, and  to  give  a  better  and  more  flocculent  precipitate  of  the  proteid. 
If  acetic  acid  is  used  for  this,  then  add  1-2-3  drops  of  a  25  per  cent  acid 
to  each  10  c.  c.  of  the  urine  and  boil  after  the  addition  of  each  drop.  On 
using  nitric  acid,  add  1-2  drops  of  the  25  per  cent  acid  to  each  cubic  centi- 
meter of  the  boiling-hot  urine. 

On  using  acetic  acid,  when  the  quantity  of  proteid  is  very  small,  and 
especially  when  the  urine  was  originally  alkaline,  the  proteid  may  some- 
times remain  in  solution  on  the  addition  of  the  above  quantity  of  acid. 
If,  on  the  contrary,  less  acid  is  added,  the  precipitate  of  calcium  phos- 
phate, which  forms  in  amphoteric  or  faintly  acid  urines,  is  liable  not 
to  dissolve  completely,  and  this  may  cause  it  to  be  mistaken  for  a  proteid 
precipitate.  If  nitric  acid  is  used  for  the  heat  test,  the  fact  must  not  be 
overlooked  that  after  the  addition  of  only  a  little  acid  a  combination  be- 
tween it  and  the  proteid  is  formed  which  is  soluble  on  boiling  and  which  is 
only  precipitated  by  an  excess  of  the  acid.  On  this  account  the  large 
quantity  of  nitric  acid,  as  suggested  above,  must  be  added,  but  in  this 
case  a  small  part  of  the  proteid  is  liable  to  be  dissolved  by  the  excess  of 
the  nitric  acid.  When  the  acid  is  added  after  boiling,  which  is  absolutely 
necessary,  the  liability  of  a  mistake  is  not  so  great.  It  is  on  these  grounds 
that  the  heat  test,  although  it  gives  very  good  results  in  the  hands  of 
experts,  is  not  recommended  to  physicians  as  a  positive  test  for  proteid. 

A  confounding  with  mucin,  when  this  body  occurs  in  the  urine,  is  easily 
prevented  in  the  heat  test  with  acetic  acid  by  acidifying  another  portion 
with  acetic  acid  at  the  ordinary  temperature.  Mucin  and  nucleoalbumin 
substances  similar  to  mucin  are  hereby  precipitated.  If  in  the  perform- 
ance of  the  heat  and  nitric-acid  test  a  precipitate  first  appears  on  cooling 
or  is  strikingly  increased,  then  this  shows  the  presence  of  albumoses  in  the 
urine,  either  alone  or  mixed  with  coagulable  proteid.  In  this  case  a  further 
investigation  is  necessary  (see  below).  In  a  urine  rich  in  urates  a  precipitate 
consisting  of  uric  acid  separates  on  cooling.  This  precipitate  is  colored  and 
granular,  and  is  hardly  to  be  mistaken  for  an  albumose  or  proteid  precipitate. 

Heller's  test  is  performed  as  follows  (see  page  30) :  The  urine  is  very 
carefully  floated  on  the  surface  of  nitric  acid  in  a  test-tube.  The  presence 
of  proteid  is  shown  by  a  white  ring  between  the  two  liquids.  With  this 
test  a  red  or  reddish-violet  transparent  ring  is  always  obtained  with  normal 
urine;  it  depends  upon  the  indigo  coloring -matters  and  can  hardly  be  mis- 
taken for  the  white  or  whitish  proteid  ring,  and  this  last  must  not  be  mis- 
taken for  the  ring  produced  by  bile-pigments.  In  a  urine  rich  in  urates 
another  complication  may  occur,  due  to  the  formation  of  a  ring  produced 
by  the  precipitation  of  uric  acid.     The  uric-acid  ring  does  not  lie,  like  the 


PROTEIDS   IN   URINE.  549 

proteid  ring;,  hot  worn  the  two  liquids,  but  somewhat  higher.  For  this  rea- 
son two  simultaneous  rings  may  exist  in  urines  which  are  rich  in  urates  and 
do  not  contain  very  much  proteid.  The  disturbance  caused  by  uric  acid 
is  easily  prevented  by  diluting  the  urine  with  1-2  vols,  of  water  before 
performing  the  test.  The  uric  acid  now  remains  in  solution,  and  the 
delicacy  of  Eelleb's  test  is  so  great  that  after  dilution  only  in  the  pres- 
ence of  insignificant  traces  of  proteid  does  this  test  give  negative  results. 
In  a  urine  very  rich  in  urea  a  ring-like  separation  of  urea  nitrate  may 
also  appear.  This  ring  consists  of  shining  crystals,  and  it  does  not  appear 
in  urine  previously  diluted.  A  confusion  with  resinous  acids,  which  also 
give  a  whitish  ring  with  this  test,  is  easily  prevented,  since  these  acids 
are  soluble  on  the  addition  of  ether.  Stir,  add  ether,  and  carefully  shake 
the  contents  of  the  test-tube.  If  the  cloudiness  is  due  to  resinous  acids, 
the  urine  gradually  becomes  clear,  and  on  evaporating  the  ether  a  sticky 
residue  of  resinous  acids  is  obtained.  A  liquid  which  contains  true  mucin 
does  not  give  a  precipitate  with  this  test,  but  it  gives  a  more  or  less  strongly 
opalescent  ring,  which  disappears  on  stirring.  The  liquid  does  not  con- 
tain any  precipitate  after  stirring,  but  is  somewhat  opalescent.  If  a  faint 
not  wholly  typical  reaction  is  obtained  with  Heller's  test  after  some 
time  with  undiluted  urine,  while  the  diluted  urine  gives  a  pronounced 
reaction,  the  presence  is  shown  of  the  substance  which  used  to  be  called 
mucin  or  nucleoalbumin.  In  this  case  proceed  as  described  below  for  the 
detection  of  nucleoalbumin. 

If  the  above-mentioned  possible  errors  and  the  means  by  which  they  may 
be  prevented  are  borne  in  mind,  there  is  hardly  another  test  for  proteid 
in  the  urine  which  is  at  the  same  time  so  easily  performed,  so  delicate,  and 
so  positive  as  Heller's.  With  this  test  even  0.002  per  cent  of  albumin 
may  be  detected  without  difficulty.  Still  the  student  must  not  be  satisfied 
with  this  test  alone,  but  should  apply  at  least  a  second  one,  such  as  the  heat 
test.     In  performing  this  test  the  (primary)  proteoses  are  also  precipitated. 

The  reaction  with  metaphosphoric  acid  (see  page  30)  is  very  convenient 
and  easily  performed.  It  is  not  quite  so  delicate  and  positive  as  Heller's 
test.     The  proteoses  are  also  precipitated  by  this  reagent. 

Reaction  with  Acetic  Acid  and  Potassium  Ferrocyanide.  Treat  the  urine 
first  with  acetic  acid  until  it  contains  about  2  per  cent,  and  then  add  drop 
by  drop  a  potassium-f errocyanide  solution  ( 1 :  20) ,  carefully  avoiding  an 
excess.  This  test  is  very  good,  and  in  the  hands  of  experts  it  is  even  more 
delicate  than  Heller's.  In  the  presence  of  very  small  quantities  of  pro- 
teid it  requires  more  practice  and  dexterity  than  Heller's,  as  the  relative 
quantities  of  reagent,  proteid,  and  acetic  acid  influence  the  result  of  the 
tesl .  The  quantity  of  salts  in  the  urine  likewise  seems  to  have  an  influence. 
This  reagent  also  precipitates  proteoses. 

Spiegler  's  Test.  Spiegler  recommends  a  solution  of  8  parts  mercuric  chloride, 
4  parts  tartaric  acid,  20  parts  glycerine,  and  200  parts  water  as  a  very  delicate 
reagent  for  proteid  in  the  urine.  A  test-tube  is  half  filled  with  this  reagent  and 
from  a  pipette  the  urine  is  allowed  to  flow  upon  its  surface  drop  by  drop  along  the 
wall  of  the  test-tube.  In  the  presence  of  proteid  a  white  ring  is  obtained  at 
the  point  of  contact  between  the  two  liquids.  The  delicacy  of  this  test  is  1 :  350000. 
Jolles  l  does  not  consider  this  reagent  suited  for  urines  very  poor  in  chlorine,  and 

'Spiegler,  Wien.  klin.  Wochensehr.,  1S92,  and  Centralbl.  f.  d.  klin.  Med.,  1893; 
Jolles,  Zeitschr.  f.  physiol.  Chem.,  21. 


550  URINE. 

for  this  reason  he  has  changed  it  as  follows:  10  grams  mercuric  chloride,  20  grams 
succinic  acid,  10  grams  NaCl,  and  500  c.  c.  water. 

Roch's  Test.  Treat  the  urine  either  with  a  20  per  cent  watery  solution  of 
sulphosalicylic  acid  or  a  few  crystals  of  the  acid.  This  reagent  does  not  precipitate 
the  uric  acid  or  the  resin  acids.1 

As  every  normal  urine  contains  traces  of  proteid,  it  is  apparent  that 
very  delicate  reagents  are  only  to  be  used  with  the  greatest  caution.  For 
ordinary  cases  Heller's  test  is  sufficiently  delicate.  If  no  reaction  is 
obtained  with  this  test  within  2\  to  3  minutes,  the  urine  tested  contains 
less  than  0.003  per  cent  of  proteid,  and  is  to  be  considered  free  from  proteid 
in  the  ordinary  sense. 

The  use  of  precipitating  reagents  presumes  that  the  urine  to  be  investi- 
gated is  perfectly  clear,  especially  in  the  presence  of  only  very  little  pro- 
teid. The  urine  must  first  be  filtered.  This  is  not  easily  done  with  urine 
containing  bacteria,  but  a  clear  urine  may  be  obtained,  as  suggested  by 
A.  Jolles,  by  shaking  the  urine  with  infusorial  earth.  Although  a  little 
proteid  is  retained  in  this  procedure  and  lost  it  does  not  seem  to  be  of  any 
importance  (Grutzner,  Schweissinger  2). 

The  different  color  reactions  cannot  be  directly  used,  especially  in  deep- 
colored  urines  which  only  contain  little  proteid.  The  common  salt  of  the 
urine  has  a  disturbing  action  on  Millon's  reagent.  To  prove  more  posi- 
tively the  presence  of  proteid,  the  precipitate  obtained  in  the  boiling  test 
may  be  filtered,  washed,  and  then  tested  with  Millon's  reagent.  The 
precipitate  may  also  be  dissolved  in  dilute  alkali  and  the  biuret  test  applied 
to  the  solution.  The  presence  of  proteoses  or  peptones  in  the  urine  is 
directly  tested  for  by  this  last-mentioned  test.  In  testing  the  urine  for 
proteid  one  should  never  be  satisfied  with  one  reaction  alone,  but  must 
apply  the  heat  test  and  Heller's  or  the  potassium-ferrocyanide  test.  In 
using  the  heat  test  alone  the  proteoses  may  be  easily  overlooked,  but  these  are 
detected,  on  the  contrary,  by  Heller's  or  the  potassium  ferrocyanide  test. 
If  only  one  of  these  tests  is  employed,  no  sufficient  intimation  of  the  kind  of 
proteid  present  can  be  obtained,  whether  it  consists  of  proteoses  or  coagu- 
lable  proteid. 

For  practical  purposes  several  dry  reagents  for  proteid  have  been  recommended. 
Besides  the  metaphosphoric  acid  may  be  mentioned  Stutz's  or  Furbringer's 
gelatine  capsules,  which  contain  mercuric  chloride,  sodium  chloride,  and  citric 
acid;  and  (Jeissler's  albumin-test  papers,  which  consist  of  strips  of  filter-paper 
which  have  been  dipped  in  a  solution  of  citric  acid  and  aisc  mercuric-chloride  and 
potassium-iodide  solution  and  then  dried. 

If  the  presence  of  proteid  has  been  positively  proved  in  the  urine  by 
the  above  tests,  it  then  remains  necessary  to  determine  its  character. 

The  Detection  of  Globulin  and  Albumin.  In  detecting  serglobulin  the 
urine  is  exactly  neutralized,  filtered,  and  treated  with  magnesium  sulphate 
in  substance  until  it  is  completely  saturated  at  the  ordinary  temperature, 
or  with  an  equal  volume  of  a  saturated  neutral  solution  of  ammonium  sul- 
phate. In  both  cases  a  white,  flocculent  precipitate  is  formed  in  the 
presence  of  globulin.  In  using  ammonium  sulphate  with  a  urine  rich  in 
urates  a  precipitate  consistine;  of  ammonium  urate  may  separate.     This 

1  Pharmaceut.  Centralhallc,  1889,  and  Zeitschr.  f.  anal.  Chem.,  29. 

2  Jolles,  Zeitschr.  f.  anal.  Chem.,  29;  Grutzner,  Chem.  Centralbl.,  1901,  1;  Schweis- 
singer, ibid. 


PROTEOSES  AND  PEPTONES.  551 

precipitate  does  not  appear  immediately,  but  only  after  a  certain  time,  and 
it  must  not  be  mistaken  for  the  globulin  precipitate.  In  detecting  ser- 
albumin heat  the  filtrate  from  the  globulin  precipitate  to  boiling-point  or 
add  about  1  per  cent  acetic  acid  to  it  at  the  ordinary  temperature. 

Proteoses  and  peptones  have  been  repeatedly  found  in  the  urine  in 
different  diseases.  Reliable  reports  are  at  hand  on  the  occurrence  of 
proteoses  in  the  urine.  The  statements  in  regard  to  the  occurrence  of 
peptones  date  in  part  from  a  time  when  the  conception  of  proteoses  and 
peptones  was  different  from  that  of  the  present  day,  and  in  part  they  are 
based  upon  investigations  using  untrustworthy  methods.  According  to 
Ito  l  true  peptones  are  sometimes  found  in  the  urine  in  cases  of  pneu- 
monia; what  has  been  designated  as  urine  peptone  seems  to  have  been 
chiefly  deuteroproteose. 

In  detecting  the  proteoses  the  proteid-free  urine,  or  urine  boiled  with 
addition  of  acetic  acid,  is  saturated  with  ammonium  sulphate,  which  precipi- 
tates the  proteoses.  Several  errors  are  here  possible.  The  urobilin,  which 
may  give  a  reaction  similar  to  the  biuret  reaction,  is  also  precipitated  and 
may  lead  to  mistakes  (Salk<  iwski,  Stokvis  2).  A  small  quantity  of  the  pro- 
teid may  remain  in  solution  after  coagulation  and  this  maybe  precipitated 
by  the  ammonium  sulphate  and  be  mistaken  for  proteoses.  The  coagu- 
lable  proteid  may  be  completely  precipitated  by  saturating  with  ammo- 
nium sulphate  in  boiling  solution;  but  according  to  Devoto  3  small  quan- 
tities of  proteose  may  be  formed  from  the  proteid  by  heating  for  a  long 
time  with  the  salt.  On  heating  for  a  short  time  no  such  formation  of 
proteose  takes  place,  and  the  proteids  are  completely  coagulated. 

For  these  reasons  Bang  *  has  suggested  the  following  method  for  the 
detection  of  proteoses  in  the  presence  of  coagulable  proteid.  The  urine  is 
heated  to  boiling  with  ammonium  sulphate  (8  parts  to  10  parts  urine) 
and  boiled  for  a  few  seconds.  The  hot  liquid  is  centrifuged  for  h  to  1  min- 
ute and  separated  from  the  sediment.  The  urobilin  is  removed  from 
this  by  extraction  with  alcohol.  The  residue  is  suspended  in  a  little  water, 
heated  to  boiling,  filtered,  whereby  the  coagulable  proteid  is  retained  on 
the  filter,  and  any  urobilin  still  present  in  the  filtrate  is  shaken  out  with 
chloroform.  The  watery  solution,  after  removal  of  the  chloroform,  is 
used  for  the  biuret  test.  For  clinical  purposes  this  method  is  very  service- 
able. 

According  to  Salkowski  the  urine  treated  with  10  per  cent  hydrochloric 
acid  is  precipitated  with  phosphotungstic  acid,  then  warmed,' the  liquid 
decanted  from  the  resin-like  precipitate,  this  washed  with  water,  and 
then  dissolved  in  a  little  water  with  the  aid  of  some  caustic  soda,  warmed 
again  until  the  blue  color  disappears,  cooled,  and  finally  tested  with  copper 


1  In  regard  to  the  literature  on  proteoses  and  peptones  in  urine,  see  Huppert- 
Neubauer,  Ham- Analyse,  10.  Aufl.,  4G6  to  492;  also  A.  Stoffregen,  Ueber  das  Vorkom- 
men  von  Pepton  nn  Harn,  Sputum  und  Eiter  (Inaug.-Diss.,  Dorpat,  1891);  E.  Hirsch- 
feldt,  Ein  Beitrag  zur  Frage  der  Peptonurie  (Inaug.-Diss.,  Dorpat,  1892);  and  espe- 
cially Stadelmann,  Untersuchungen  iiber  die  Peptonurie.  Wiesbaden,  1894 ;  Erhstrom, 
Bidrag  till  k-innedomen  om  AJbumosurien,  Helsingfors,  1900;  Ito,  DeUtsch  Arch. 
f.  klin.  Med.,  71. 

'Salkowski,  Berlin,  klin.  Wochenschr.,  1897;  Stokvis,  Zeitschr.  f.  Biologie,  34. 

s  Zeitschr.  f.  physiol.  Chem.,  15. 

*Deutsch.  med.  Wochenschr.,  1898. 


552  URINE. 

sulphate.  This  method  has  been  recently  somewhat  modified  by  v.  Aldor 
and  Cerny.1  In  regard  to  other  more  complicated  methods  we  refer  to- 
Huppert-Neubauer. 

If  the  proteoses  have  been  precipitated  from  a  larger  portion  of  urine 
by  ammonium  sulphate,  this  precipitate  is  tested  for  the  presence  of  dif- 
ferent proteoses  for  the  reasons  given  in  Chapter  II.  The  following  serves' 
as  a  preliminary  determination  of  the  character  of  the  proteoses  present 
in  the  urine.  If  the  urine  contains  only  deuteroproteose  it  does  not  become 
cloudy  on  boiling,  does  not  give  Heller's  test,  does  not  become  cloudy 
on  saturating  with  NaCl  in  neutral  reaction,  but  does  become  turbid  on 
adding  acetic  acid  saturated  with  this  salt.  In  the  presence  of  only  proto- 
proteose  the  urine  gives  Heller's  test,  is  precipitated  even  in  neutral 
solution  on  saturating  with  NaCl,  but  does  not  coagulate  on  boiling.  The 
presence  of  heteroproteose  is  shown  by  the  urine  behaving  like  the  above 
with  NaCl  and  nitric  acid,  but  shows  a  difference  on  heating.  It  gradually 
becomes  cloudy  on  warming  and  separates  at  about  60°  C.  a  sticky  precipi- 
tate which  attaches  itself  to  the  sides  of  the  vessel  and  which  dissolves  at 
boiling  temperature  on  acidifying  the  urine;  the  precipitate  reappears  on 
cooling. 

In  close  relation  to  the  proteoses  stands  the  so-called  Bence-Jones 
proteid,  which  occurs  in  the  urine  in  rare  cases  in  disease  with  changes  in 
the  spinal-marrow.  It  gives  a  precipitate  on  heating  to  40-60°  C,  which  on 
further  heating  to  boiling  dissolves  again  more  or  less  completely,  depending 
upon  the  reaction  and  upon  the  amount  of  salt  present.  It  does  not  sepa- 
rate on  dialysis,  but  can  be  precipitated  from  the  urine  by  double  the 
volume  of  a  saturated  ammonium-sulphate  solution  or  by  alcohol.  It 
has  also  been  obtained  as  crystals  (Grutterink  and  de  Graaff,  Magnus- 
Levy  2).  This  body  shows  a  somewhat  different  behavior  in  the  various 
cases  in  which  it  has  been  found  and  its  nature  has  not  been  explained. 

Quantitative  Estimation  of  Proteid  in  Urine.  Of  all  the  methods  pro- 
posed thus  far,  the  coagulation  method  (boiling  with  the  addition  of 
acetic  acid)  when  performed  with  sufficient  care  gives  the  best  results. 
The  average  error  need  never  amount  to  more  than  0.01  per  cent,  and  it 
is  generally  smaller.  In  using  this  method  it  is  best  to  first  find  how  much 
acetic  acid  must  be  added  to  a  small  portion  of  the  urine,  which  has  been 
previously  heated  on  the  water-bath,  to  completely  separate  the  proteid  so 
that  the  filtrate  does  not  respond  to  Heller's  test.  Then  coagulate 
20-50-100  c.  c.  of  the  urine.  Pour  the  urine  into  a  beaker  and  heat  on 
the  water-bath,  add  the  required  quantity  of  acetic  acid  slowly,  stirring 
constantly,  and  heat  at  the  same  time.  Filter  while  warm,  wash  first  with 
water,  then  with  alcohol  and  ether,  dry  and  weigh,  incinerate  and  weigh 
again.     In  exact  determinations  the  filtrate  must  not  give  Heller's  test. 

The  separate  estimation  of  globulins  and  albumins  is  done  by  care- 
fully neutralizing  the  urine  and  precipitating  with  MgS04  added  to  satura- 
tion (Hammarsten),  or  simply  by  adding  an  equal  volume  of  a  saturated 
neutral  solution  of  ammonium  sulphate  (Hofmeister  and  Pohl3).    The 

1  Salkowski,  Centralbl.  f.  d.  med.  Wissensch.,  1894;  v.  Aldor,  Berl.  klin.  Wochenschr., 
30;   Cerny,  Zeitschr.  f.  analyt.  Chem.,  40. 

2  Magnus-Levy,  Zeitschr.  f.  physiol.  Chem.,  30  fliterature);  Grutterink  and  de 
Graaff.  ibid.,  34. 

3  Hammarsten,  Pfliiger's  Arch.,  17;  Hofmeister  and  Pohl,  Arch.  f.  exp.  Path.  u. 
Pharm.,  20. 


NUCLEOALBUMIN  AND  MUCIN.  553 

precipitate  consisting  of  globulin  is  thoroughly  washed  with  a  saturated 
tesium-sulphate  or  half-saturated  ammonium-sulphate  solution,  dried 

continuously    at    110°  (\,    boiled    with    Water,    extracted    with    alcohol    and 

ether,  then' dried,  weighed,  incinerated,  and  weighed  again.  The  quan- 
tity of  albumin  is  calculated  as  the  difference  between  the  quantity  of 
globulin   and    the    total    proteids. 

Approximate  Estimation  of  Protcid  in  Urine.  Of  the  methods  sug- 
gested for  this  purpose  none  has  been  more  extensively  employed  than 
Esbach's. 

Esbach's1  Method.  The  acidified  urine  (with  acetic  acid)  is  pound 
into  a  specially  graduated  tube  to  a  certain  mark,  and  then  the 
reagent  (a  2  per  cent  citric-acid  and  1  per  cent  picric-acid  solution  in  water) 
is  added  to  a  second  mark,  the  tube  closed  with  a  rubber  stopper  and  care- 
fully shaken,  avoiding  the  production  of  froth.  The  tube  is  allowed  to 
stand  twenty-four  hours,  and  then  the  height  of  the  precipitate  on  the 
graduation  is  read  off.  The  reading  gives  directly  the  quantity  of 
proteid  in  1000  parts  of  the  urine.  Urines  rich  in  proteid  must  first  be 
diluted  with  water.  The  results  obtained  by  this  method  are,  however, 
dependent  upon  the  temperature;  and  a  difference  in  temperature  of  5°  to 
6.5°  C.  may  cause  an  error  of  0.2-0.3  per  cent  deficiency  or  excess  in  urines 
containing  a  medium  quantity  of  proteid  (Christensen  and  Mygge  *). 
This  method  is  only  to  be  used  in  a  room  in  which  the  temperature  may  be 
kept  nearly  constant.     The  directions  for  its  use  accompany  the  apparatus. 

Other  methods  for  the  approximate  estimation  of  proteid  are  the  optical 
methods  of  Christensen  and  Mygge,  of  Roberts  and  Stolnikow  as  modified 
by  Brandberg,  with  Heller's  test,  which  has  been  simplified  for  practical 
purposes  by  Mittelbach  The  density  methods  of  Lang,  Huppert  and  Zahor 
are  also  very  good.  In  regard  to  these  and  other  methods  we  refer  to  Huppert- 
Neubauer's  Harn-Analyse,  10.  Aufl. 

There  is  at  present  no  trustworthy  method  for  the  quantitative  estimation 
of  proteoses  and  peptone  in  the  urine. 

Nucleoalbumin  and  Mucin.  According  to  K.  Morner  traces  of  urinary 
mucoids  may  pass  into  solution  in  the  urine;  otherwise  normal  urine 
contains  no  mucin.  There  is  no  doubt  that  there  may  be  cases  where  true 
mucin  appears  in  the  urine;  in  most  cases  mucin  has  probably  been  mis- 
taken for  so-called  nucleoalbumin.  The  occurrence,  under  some  circum- 
stances, of  nucleoalbumin  in  the  urine  is  not  to  be  denied,  as  such  substances 
occur  in  the  renal  and  urinary  passages;  still  in  most  cases  this  nucleo- 
albumin, as  shown  by  K.  Morner,8  is  of  an  entirely  different  kind. 

Every  urine,  according  to  Morner,  contains  a  little  proteid  and  in 
addition  substances  precipitating  proteid.  If  the  urine  freed  from  salts  by 
dialysis  is  shaken  with  chloroform  after  the  addition  of  1-2  p.  m.  acetic 
acid,  a  precipitate  is  obtained  which  acts  like  a  nucleoalbumin.  If  the 
acid  filtrate  is  treated  with  seralbumin,  a  new  and  similar  precipitate  is 

1  In  regard  to  the  literature  on  this  method  and  the  numerous  experiments  to  deter- 
mine its  value,  see  Huppert-Neubauer,  10.  Aufl.,  853. 

2  Christensen,  Virchow's  Arch.,  115. 

3  Skand.  Arch.  f.  Physiol.,  0. 


554  URINE. 

obtained  due  to  the  presence  of  a  residue  of  the  substance  which  precipi- 
tates proteids.  The  most  important  of  these  proteid-precipitating  sub- 
stances is  chondroitin-sulphuric  acid  and  nucleic  acid,  although  the  latter 
appears  to  a  much  smaller  extent.  Taurocholic  acid  may  in  a  few  instances, 
especially  in  icteric  urines,  be  precipitated.  The  substances  isolated  by 
different  investigators  from  urine  by  the  addition  of  acetic  acid  and  called 
"dissolved  mucin"  or  "nucleoalbumin"  are  considered  by  Morner  to  be 
a  combination  of  proteid  with  chondroitin-sulphuric  acid  chiefly,  and  to  a 
less  extent  with  nucleic  acid,  and  also  perhaps  with  taurocholic  acid. 

As  normal  urine  habitually  contains  an  excess  of  substances  capable  of 
precipitating  proteids,  it  is  apparent  that  an  increased  elimination  of  so- 
called  nucleoalbumin  may  be  caused  simply  by  an  augmented  excretion  of 
proteid.  This  happens  to  a  still  greater  extent  in  cases  where  the  proteid 
as  well  as  the  proteid-precipitating  substance  is  eliminated  to  an  increased 
extent. 

Detection  of  so-called  Nucleoalbumins.  When  a  urine  becomes  cloudy 
or  precipitates  on  the  addition  of  acetic  acid,  and  when  it  gives  a  more 
typical  reaction  with  Heller  's  test  after  the  dilution  of  the  urine  than  before, 
one  is  justified  in  making  tests  for  mucin  and  nucleoalbumin.  As  the  salts 
of '  the  urine  interfere  considerably  with  the  precipitation  of  these  sub- 
stances by  acetic  acid,  they  must  first  be  removed  by  dialysis.  As  large 
a  quantity  of  urine  as  possible  is  dialyzed  (with  the  addition  of  chloroform) 
until  the  salts  are  removed.  Then  acetic  acid  is  added  until  it  contains 
2  p.  m.,  and  the  mixture  allowed  to  stand.  The  precipitate  is  dissolved  in 
water  by  the  aid  of  the  smallest  possible  quantity  of  alkali  and  precipitated 
again.  In  testing  for  chondroitin-sulphuric  acid  a  part  is  warmed  on  the 
water-bath  with  about  5  per  cent  hydrochloric  acid.  If  positive  results  are 
obtained  on  testing  for  sulphuric  acid  and  a  reducing  substance,  then  chon- 
droproteid  was  present.  If  a  reducing  substance  can  be  detected  but  no 
sulphuric  acid,  then  mucin  is  probably  there.  If  it  does  not  contain  any 
sulphuric  acid  or  reducing  substance,  a  part  of  the  precipitate  is  exposed 
to  pepsin  digestion  and  another  part  used  for  the  determination  of  any 
organic  phosphorus.  If  positive  results  are  obtained  from  these  tests, 
then  nucleoalbumin  and  nucleoproteid  must  be  differentiated  by  special 
tests  for  nuclein  bases.  No  positive  conclusion  can  be  drawn  except 
by  using  very  large  quantities  of  urine. 

Nucleohiston.  In  a  case  of  pseudole  ca?mia  A.  Jolles  found  a  phosphorized 
protein  substance  which  he  considers  as  identical  with  nucleohiston.  Histon  is 
claimed  to  have  been  found  in  some  cases  by  Krehl  ;md  Matthes  and  by  Kolisch 
and  Burian.1 

Blood  and  Blood-coloring  Matters.  The  urine  may  contain  blood  from 
hemorrhage  in  the  kidneys  or  other  parts  of  the  urinary  passages  (hema- 
turia). In  these  cases,  when  the  quantity  of  blood  is  not  very  small,  the 
urine  is  more  or  less  cloudy  and  colored,  reddish,  yellowish-red,  dirty  red, 
brownish-red,  or  dark  brown.     In  recent  hemorrhages,  in  which  the  blood 

1  Jolles,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30;  Krehl  and  Matthes,  Deutsch.  Arch, 
f.  klin.  Med.,  54;  Kolisch  and  Burian,  Zeitschr.  f.  klin.  Med.,  29. 


BLOOD  IN  THE   URINE. 

has  not  decomposed,  the  color  is  nearer  blood-red.     Blood-corpuscles  may 

be    found  in  the  sediment,  sometimes  also  blood-casts  and  smaller  or  larger 

blood-clots. 

In  certain  cases  the  urine  contains  no  blood-corpuscles,  but  only  dis- 
solved blood-coloring  matters,  haemoglobin,  or,  and  indeed  quite  often, 
methsemoglobin  (hemoglobinuria).  The  blood-pigmenta  appeal-  in  the 
urine  under  different  conditions,  as  in  dissolution  of  blood  in  poisoning  with 
arseniuretted  hydrogen,  chlorates,  etc.,  after  serious  burns,  after  trans- 
fusion of  blood,  and  also  in  the  periodic  appearance  of  hemoglobinuria 
with  fever.  In  nsemoglobinuria  the  urine  may  also  have  an  abundant 
grayish-brown  sediment  rich  in  proteid  which  contains  the  remains  of  the 
stromata  of  the  red  blood-corpuscles.  In  animals  nsemoglobinuria  may 
be  produced  by  many  causes  which  force  free  haemoglobin  into  the  plasma. 

To  detect  blood  in  the  urine  we  make  use  of  the  microscope,  spectro- 
scope, the  guaiacum  test,  and  Heller's  or  Heller-Tkich.mann's  test. 

Microscopic  Investigation.  The  blood-corpuscles  may  remain  undis- 
solved for  a  long  time  in  acid  urine;  in  alkaline  urine,  on  the  contrary,  they 
are  easily  changed  and  dissolved.  They  often  appear  entirely  unchanged  in 
the  sediment;  in  some  cases  they  are  distended  and  in  others  unequally 
pointed  or  jagged  like  a  thorn-apple.  In  hemorrhage  of  the  kidneys  a 
cylindrical  clot  is  sometimes  found  in  the  sediment  which  is  covered  with 
numerous  red  blood-corpuscles,  forming  casts  of  the  urinary  passages. 
These  formations  are  called  blood-casts. 

The  spectroscopic  investigation  is  naturally  of  very  great  value;  and  if 
it  be  necessary  to  determine  not  only  the  presence  but  also  the  kind  of 
coloring-matter,  this  method  is  indispensable.  In  regard  to  the  optical 
behavior  of  the  various  blood-pigments  we  must  refer  to  Chapter  VI. 

Guaiacum  Test.  Mix  in  a  test-tube  equal  volumes  of  tincture  of  guaia- 
cum and  old  turpentine  which  has  become  strongly  ozonized  by  the  action 
of  air  under  the  influence  of  light.  To  this  mixture,  which  must  not  have 
the  slightest  blue  color,  add  the  urine  to  be  tested.  In  the  presence  of  blood 
or  blood-pigments,  first  a  bluish-green  and  then  a  beautiful  blue  ring 
appears  where  the  two  liquids  meet.  On  shaking  the  mixture  it  becomes 
more  or  less  blue.  Normal  urine  or  one  containing  proteid  does  not  give 
this  reaction.  For  the  explanation  of  this  we  must  refer  the  reader  to 
Chapter  VI,  page  169.  Urine  containing  pus,  although  no  blood  is  present, 
gives  a  blue  color  with  these  reagents;  but  in  this  case  the  tincture  of 
guaiacum  alone,  without  turpentine,  is  colored  blue  by  the  urine  (Vitali  l). 
This  is  at  least  true  for  a  tincture  that  has  been  exposed  for  some  time 
to  the  action  of  air  and  sunlight.  The  blue  color  produced  by  pus  differs 
from  that  produced  by  blood-coloring  matters  by  disappearing  on  heat  inn 
the  urine  to  boiling.  A  urine  alkaline  by  decomposition  must  first  be 
made  faintly  acid  before  performing  the  reaction.     The  turpentine  should 

1  See  Maly's  Jahresber.,  18. 


556  URINE. 

be  kept  exposed  to  sunlight,  while  the  tincture  of  guaiacum  must  be  kept 
in  a  dark  glass  bottle.  These  reagents  to  be  of  use  must  be  controlled 
by  a  liquid  containing  blood.  This  test,  it  is  true,  with  positive  results  is 
not  absolutely  decisive,  because  other  bodies  may  give  a  similar  reaction; 
but  when  properly  performed  it  is  so  extremely  delicate  that  when  it  gives 
negative  results  any  other  test  for  blood  is  superfluous. 

Heller-Teichmann's  Test.  If  a  neutral  or  faintly  acid  urine  contain- 
ing blocd  is  heated  to  boiling,  one  always  obtains  a  mottled  precipitate 
consisting  of  proteid  and  hsematin.  If  caustic  soda  is  added  to  the  boiling- 
hot  test,  the  liquid  becomes  clear  and  turns  green  when  examined  in  thin 
layers  (due  to  hsematin  alkali),  and  a  red  precipitate,  appearing  green  by 
reflected  light,  re-forms,  consisting  of  earthy  phosphates  and  haematin. 
This  reaction  is  called  Heller's  blood-test.  If  this  precipitate  is  col- 
lected after  a  time  on  a  small  filter,  it  may  be  used  for  the  hsemin  test 
(see  pa°;e  179).  If  the  precipitate  contains  only  a  little  blood-coloring  mat- 
ter with  a  larger  quantity  of  earthy  phosphates,  then  wash  it  with  dilute 
acetic  acid,  which  dissolves  the  earthy  phosphates,  and  use  the  residue  for 
the  preparation  of  Teichmann's  haemin  crystals.  If,  on  the  contrary,  the 
amount  of  phosphates  is  very  small,  then  first  add  a  little  CaCl2  solution  to 
the  urine,  heat  to  boiling,  and  add  simultaneously  with  the  caustic  potash 
some  sodium-phosphate  solution.  In  the  presence  of  only  very  small 
quantities  of  blood,  first  make  the  urine  very  faintly  alkaline  with  am- 
monia, add  tannic  acid,  acidify  with  acetic  acid,  and  use  this  precipitate  in 
the  preparation  of  the  hsemin  crystals  (Struve  1). 

Hasmatoporphyrin.  Since  the  occurrence  of  hsematoporphyrin  in  the 
urine  in  various  diseases  has  been  made  very  probable  by  several  investi- 
gators, such  as  Neusser,  Stokvis,  MacMuxn,  Le  Nobel,  Russel,  Cope- 
man,  and  others,2  Salkowski  has  positively  shown  the  presence  of  this 
pigment  in  the  urine  after  sulphonal  intoxication.  It  was  first  isolated 
in  a  pure  crystalline  state  b}^  Hammarsten  3  from  the  urine  of  insane 
women  after  sulphonal  intoxication.  According  to  Garrod  and  Saillet  4 
traces  of  hasmatoporphyrin  (Saillet's  urospectrin)  occur  regularly  in 
normal  urines.  It  is  also  found  in  the  urine  during  different  diseases, 
although  it  only  occurs  in  small  quantities.  It  has  been  found  in  consider- 
able quantities  in  the  urine  after  the  lengthy  use  of  sulphonal. 

Urine  containing  hsematoporphyrin  is  sometimes  only  slightly  colored, 
while  in  other  cases,  as  for  example  after  the  use  of  sulphonal,  it  is  more  or 
less  deep  red.  The  color  depends  in  these  last-mentioned  cases,  in  greatest 
part,  not  upon  the  hsematoporphyrin,  but  upon  other  red  or  reddish- 
brown  pigments  which  have  not  been  sufficiently  studied. 

1  Zeitschr.  f.  anal.  Chem.,  11. 

2  A  very  complete  index  of  the  literature  on  hiumatoporphyrin  in  the  urine  may  be 
found  in  It.  Zoja,  Su  qualche  pigmento  di  alcune  urine,  etc.,  in  Arch.  Ital.  di.  clin. 
Med.,  1893. 

3  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  15;  Hammarsten,  Skand.  Arch.  f.  Physiol.,  3 

4  Garrod,  Journ.  of  Physiol.,  13  (contains  review  of  literature)  and  17;  Saillet, 
Ptevue  de  mtfdecine,  16. 


H.EMATOPORPHYRIN.     MELANIN,  557 

In  the  detection  of  small  quantities  of  hsematopoiphyrin  proceed  a3 
suggested  by  (  Iabrod.     Precipitate  the  urine  with  a  10  per  cent  caustic-soda 

solution  (20  c.  c.  for  every  100  c.  c.  of  urine).  The  phosphate  precipitate 
containing  the  pigment  is  dissolved  in  alcohol-hydrochloric  acid  (15-20  c.  c.) 
and  the  solution  investigated  by  the  spectroscope.  In  more  exact  inves- 
tigations make  the  solution  alkaline  with  ammonia,  ad< I  enough  acetic  acid 
to  dissolve  the  phosphate  precipitate,  shake  with  chloroform,  which  takes 
up  the  pigment,  and  test  this  solution  with  the  spectroscope. 

In  the  presence  of  larger  quantities  of  haematoporphyrin  the  urirjj 
first  precipitated,  according  to  Salkowski,  with  an  alkaline  barium- 
chloride  solution  (a  mixture  of  equal  volumes  of  barium-hydrate  solution, 
saturated  in  the  cold,  and  a  10  per  cent  barium-chloride  solution),  or,  accord- 
ing to  HamMAKSTEN,1  with  a  barium-acetate  solution.  The  washed  pre- 
cipitate, which  contains  the  haematoporphyrin,  is  allowed  to  stand  some 
time  at  the  temperature  of  the  room  with  alcohol  containing  hydrochloric 
or  sulphuric  acid  and  then  filtered.  The  filtrate  shows  the  characteristic 
spectrum  of  haematoporphyrin  in  acid  solution  and  gives  the  spectrum 
of  alkaline  hannatoporphyrin  after  saturation  with  ammonia.  If  the 
alcoholic  solution  is  mixed  with  chloroform  and  a  large  quantity  of  water 
added  and  carefully  shaken,  sometimes  a  lower  layer  of  chloroform  is 
obtained  which  contains  very  pure  haematoporphyrin.  while  the  upper 
layer  of  alcohol  and  water  contains  the  other  pigments  besides  some  haema- 
toporphyrin. 

Other  methods  which  have  no  advantage  over  this  one  of  Garrod  have  been 
suggested  by  Riva  and  Zoja  as  well  as  Saillet.2 

Baumstark  3  found  in  a  case  of  leprosy  two  characteristic  coloring-matters 
in  the  urine,  '  urorubrohsematin"  and  "urofuseohamiatin,"  which,  as  their  names 
indicate,  seem  to  stand  in  close  relationship  to  the  blood-coloring  matters.  Uro- 
rubruhnrivitin,  CtsH9^sFe202(i,  contains  iron  and  shows  in  acid  solution  an  absorp- 
tion-band in  front  of  D  and  a  broader  one  back  of  D.  In  alkaline  solution  it 
shows  four  bands — behind  D,  at  E,  beyond  F,  and  behind  G.  It  is  not  soluble 
either  in  water,  alcohol,  ether,  or  chloroform.  It  gives  a 'beautiful  brownish-red 
non-dichroitic  liquid  with  alkalies.  Urofuscoharmatin,  CesHi0tSaO26,  which  is  free 
from  iron,  shows  no  characteristic  spectrum;  it  dissolves  in  alkalies,  producing 
a  brown  color.  It  remains  to  be  proved  whether  these  two  pigments  are  related 
to  (impure)  haematoporphyrin. 

Melanin.  In  the  presence  of  me'anotic  cancers  dark  pigments  are  some- 
times eliminated  with  the  urine.  K.  Morner  has  isolated  two  pigments  from  such 
a  urine,  of  which  one  was  soluble  in  warm  50-75  per  cent  acetic  acid  and  the 
other,  on  the  contrary,  was  insoluble.  The  one  seemed  to  be  phymatvrhusin  (see 
Chapter  XVI).  Usually  the  urine  does  net  contain  any  melanin,  but  a  chromo- 
gen  of  melanin,  a  mdamogen.  In  such  cases  the  urine  gives  Eislet's  reaction, 
becoming  dark-colored  with  oxidizing  agents  such  as  concentrated  nitric  acid, 
p  ita>sium  bichromate  and  sulphuric  acid,  as  well  as  with  free  sulphuric  acid. 
Urine  containing  melanin  or  melanogen  is  colored  black  by  a  ferric-chloride 
soluton  (v.  Jaksch  *). 

Urorosein,  so  named  by  Xencki,5  is  a  urinary  coloring-matter   occurring   in 

'Salkowski,  1.  c. ;    Hammarsten.  1.  c. 

1  Riva  and  Zoja,  Maly's  Jahresber.,  2-1;  Saillet,  1.  c.     See  also  Xebelthau,  Zeitschr. 
f.  physiol.  Chem.,  27. 
s  Pfliiger's  Arch.,  9. 

4  K.  Morner,  Zeitschr.  f.  physiol.  Chem.,  11;   v.  Jaksch,  ibid.,  13. 

5  Xencki  and  Sieber,  Journ.  f.  prakt   Chem.  (X.  P.),  2t'». 


558  URINE. 

various  diseases,  but  which  is  not  a  constituent  of  normal  urine.  The  pigment 
does  not  occur  preformed  in  the  urine,  but  first  makes  its  appearance  after  the 
addition  of  mineral  acids.  It  is  readily  soluble  in  water,  dilute  mineral  acids,  ethyl 
and  amyl  alcohol,  and  can  be  removed  from  the  acid  urine  by  shaking  with  the 
latter.  It  differs  from  indigo  red  in  the  following:  Alkalies  immediately  decolor- 
ize a  urorosein  solution,  but  not  an  indigo-red  solution.  Urorosein  is  removed 
from  its  amyl-alcohol  solution  by  shaking  with  dilute  alkali,  while  indigo  red  is  not. 
If  the  acid  urine  is  shaken  with  chloroform,  indigo  red  is  taken  up,  but  not  uroro- 
sein. Urorosein  is  soon  decomposed  by  light  and  shows  a  sharply  defined  absorp- 
tion-band between  D  and  E.  The  red  pigment  appearing  in  urines  rich  in  skatol 
after  the  addition  of  hydrochloric  acid  differs  from  urorosein  by  being  insoluble 
in  water,  but  readily  soluble  in  ether  and  chloroform.  The  statements  in 
regard  to  the  properties  of  skatol  red  are  somewhat  divergent  and  it  is  there- 
fore difficult  to  state  a  positive  difference  between  urorosein  and  skatol  red. 

Pus  occurs  in  the  urine  in  different  inflammatory  affections,  especially 
in  catarrh  of  the  bladder  and  in  inflammation  of  the  pelvis  of  the  kidneys 
or  the  urethra. 

Pus  is  best  detected  by  means  of  the  microscope.  The  pus-cells  are 
rather  easily  destroyed  in  alkaline  urines.  In  detecting  pus  we  make  use 
of  Donne's  pus  test,  which  is  performed  in  the  following  way:  Pour 
off  the  urine  from  the  sediment  as  carefully  as  possible,  place  a  small  piece  of 
caustic  alkali  on  the  sediment,  and  stir.  If  the  pus-cells  have  not  been 
previously  changed,  the  sediment  is  converted  by  this  means  into  a  slimy 
tough  mass. 

The  pus-corpuscles  swell  up  in  alkaline  urines,  dissolve,  or  at  least  are 
so  changed  that  they  cannot  be  recognized  under  the  microscope.  The 
urine  in  these  cases  is  more  or  less  slimy  or  fibrous,  and  the  proteid  can  be 
precipitated  in  large  flakes  by  acetic  acid,  so  that  it  might  possibly  be  mis- 
taken for  mucin.  The  closer  investigation  of  the  precipitate  produced  b}r 
acetic  acid,  and  especially  the  appearance  or  non-appearance  of  a  reducing 
.substance  after  boiling  it  with  a  mineral  acid,  demonstrates  the  nature  of 
the  precipitated  substance.     Urine  containing  pus  always  contains  proteid. 

Bile-acids.  The  reports  in  regard  to  the  occurrence  of  bile-acids  in  the 
urine  under  physiological  conditions  do  not  agree.  According  to  Dragen- 
dorff  and  Hone  traces  of  bile-acids  occur  in  the  urine;  according  to 
Mackay  and  v.  Udranszky  and  K.  Morner  *  they  do  not.  Pathologically 
they  are  present  in  the  urine  in  hepatogenic  icterus,  although  not  invariably. 

Detection  of  Bile-acids  in  the  Urine.  Pettenkoper 's  test  gives  the 
most  decisive  reaction;  but  as  it  gives  similar  color  reactions  with  other 
bodies,  it  must  be  supplemented  by  the  spectroscopic  investigation.  The 
direct  test  for  bile-acids  is  easily  performed  after  the  addition  of  traces  of 
bile  to  a  normal  urine.  But  the  direct  detection  in  a  colored  icteric  urine 
is  more  difficult  and  gives  very  misleading  results;  the  bile-acid  must  there- 
fore always  be  isolated  from  the  urine.  This  may  be  done  by  the  following 
method  of  Hoppe-Seyler,  which  is  slightly  modified  in  non-essential  points. 

Hoppe-Seyler 's  Method.  Concentrate  the  urine  and  extract  the 
residue  with  strong  alcohol.  The  filtrate  is  freed  from  alcohol  by  evap- 
oration  and  then  precipitated  by  basic  lead  acetate  and  ammonia.     The 

1  Cited  from  Huppert-Neubauer,  Harn-Analyse,  10.  Aufl.,  229. 


BILE-PIGMENTS  IN  THE   URINE.  559 

washed  precipitate  is  treated  with  boiling  alcohol,  filtered  hot,  the  filtrate 
treated  with  a  few  drops  of  soda  solution,  and  evaporated  to  dryness.  The 
dry  residue  is  extracted  with  absolute  alcohol,  filtered,  and  an  excess  of 
ether  added.  The  amorphous  or,  after  a  longer  time,  crystalline  precipi- 
tate consisting  of  the  alkali  salts  of  the  biliary  acids  is  used  in  performing 
Pbttbnkofeb  s  test. 

Haycbaft  has  suggested  a  reaction  for  clinical  purposes  which  consists  in 
sprinkling  flowers  of  sulphur  upon  the  urine.  In  icteric  urine  the  powder  quickly 
sinks  to  the  bottom,  while  in  normal  urine  it  remains  on  the  surface.  The  value 
of  this  test  is  still  questioned. 

Bile-pigments  occur  in  the  urine  in  different  forms  of  icterus.  A 
urine  containing  bile-pigments  is  always  abnormally  colored — yellow, 
yellowish  brown,  deep  brown,  greenish  yellow,  greenish  brown,  or  nearly 
pure  green.  On  shaking  it  froths  and  the  bubbles  are  yellow  or  yellowish 
green  in  color.  As  a  rule  icteric  urine  is  somewhat  cloudy,  and  the  sedi- 
ment is  frequently,  especially  when  it  contains  epithelium-cells,  rather 
strongly  colored  by  the  bile-pigments.  In  regard  to  the  occurrence  of 
urobilin  in  icteric  urine  see  p.  517. 

Detection  of  Bile-coloring  Matters  in  Urine.  Many  tests  have  been  pro- 
posed for  the  detection  of  these  substances.  Ordinarily  we  obtain  the 
best  results  either  with  Gmelin's  or  with  Huppert's  test. 

Gmelin's  test  may  be  applied  directly  to  the  urine;  but  it  is  better  to 
use  Rosenbach's  modification.  Filter  the  urine  through  a  very  small  filter, 
which  becomes  deeply  colored  from  the  retained  epithelium-cells  and  bodies 
of  that  nature.  After  the  liquid  has  entirely  passed  through  apply  to  the 
inside  of  the  filter  a  drop  of  nitric  acid  wdiich  contains  only  very  little 
nitrous  acid.  A  pale-yellow  spot  will  be  formed  which  is  surrounded  by 
colored  rings  which  appear  yellowish  red,  violet,  blue,  and  green  from 
within  outward.  This  modification  is  very  delicate,  and  it  is  hardly  possi- 
ble to  mistake  indican  and  other  coloring-matters  for  the  bile-pigments. 
Several  other  modifications  of  Gmelin's  direct  test,  e.g.,  with  concentrated 
sulphuric  acid,  nitrate,  etc.,  have  been  proposed,  but  they  are  neither 
simpler  nor  more  delicate  than  Rosenbach's  modification. 

Huppert's  Reaction.  In  a  dark-colored  urine  or  one  rich  in  indican 
good  results  are  not  always  obtained  with  Gmelin's  test.  In  such  cases, 
as  also  in  urines  containing  blood-coloring  matters  at  the  same  time,  the 
urine  is  treated  with  lime-water,  or  first  with  some  CaCl,  solution,  and  then 
with  a  solution  of  soda  or  ammonium  carbonate.  The  precipitate  which 
contains  the  bile-coloring  matters  is  filtered,  washed,  dissolved  in  alcohol 
which  contains  5  c.c.  of  concentrated  hydrochloric  acid  in  100  e.  C.  (I.  Mink), 
and  heated  to  boiling  when  the  solution  becomes  green  or  bluish  green. 
According  to  Nakayama  l  this  reaction  is  more  delicate  on  using  a  mix- 
ture of  ferric  chloride,  acid,  and  alcohol. 

Hammarsten's  Reaction.  For  ordinary  cases  it  is  sufficient  to  add  a  few 
drops  of  urine  to  about  2-3  c.  c.  of  the  reagent  (see  page  271s),  when  the 

'Munk,  Du  Bois-Reymond's  Arch.,  1S0.S;  Nakayama,  Zoitschr.  f.  physiol.  Chom., 
3G. 


560  URINE. 

mixture  immediately  after  shaking  turns  a  beautiful  green  or  bluish  green, 
which  color  remains  for  several  days.  In  the  presence  of  only  very  small 
quantities  of  bile-pigments,  especially  when  blood  or  other  pigments  are 
simultaneously  present,  pour  about  10  c.  c.  of  the  acid  or  nearly  neutral 
(not  alkaline)  urine  into  the  tube  of  a  small  centrifugal  machine  and  add 
BaCl2  solution  and  centrifuge  for  about  one  minute.  The  liquid  is  decanted 
off  and  the  sediment  stirred  with  about  1  c.  c.  of  the  reagent  and  centri- 
fuged  again.  A  beautiful  green  solution  is  obtained,  which  maj'  be  changed 
by  the  addition  of  increased  quantities  of  the  acid  mixture  to  blue,  violet, 
red,  and  reddish  yellow.  The  green  color  may  be  obtained  in  the  presence 
of  1  part  bile-pigment  in  500,000-1,000,000  parts  urine.  In  the  presence 
of  large  amounts  of  other  pigments  calcium  chloride  is  better  suited  than 
barium  chloride. 

Bouma  1  has  suggested  the  use  of  alcohol  containing  ferric  chloride 
and  hydrochloric  acid  instead  of  the  above-mentioned  acid  mixture. 

The  very  delicate  reaction  as  suggested  by  Jolles  is  unfortunately  not 
serviceable  on  account  of  the  formation  of  froth,  especially  in  the  presence 
of  proteid  and  blood-pigments;  but  he  has  changed  it  by  centrifuging  the 
urine  with  chloroform  and  barium  chloride  and  suspending  the  chloroform- 
barium  residue  in  alcohol;  after  which  he  treats  it  with  a  solution  of  iodine 
and  mercuric  chloride  in  alcohol  containing  hydrochloric  acid.2  The  color 
becomes  green  or  bluish  green.     This  test  seems  to  be  good. 

Stokvis's  reaction  is  especially  valuable  as  a  control  test  in  those  cases 
in  which  the  urine  contains  only  very  little  bile-coloring  matter  together 
with  larger  quantities  of  other  coloring-matters.  The  test  is  performed 
as  follows:  20-30  c.  c.  of  urine  is  treated  with  5-10  c.  c.  of  a  solution  of  zinc 
acetate  (1:5).  The  precipitate  is  washed  on  a  small  filter  with  water  and 
then  dissolved  in  a  little  ammonia.  The  new  filtrate  gives,  either  directly 
or  after  it  has  stood  a  short  time  in  the  air  until  it  has  a  peculiar  brownish- 
green  color,  the  absorption-bands  of  bilicyanin  (see  page  272).  This  reac- 
tion is  unfortunately  not  sufficiently  delicate. 

Many  other  reactions  for  bile-coloring  matters  in  the  urine  have  been 
proposed;  but  as  those  above  mentioned  are  sufficient,  it  is  perhaps  only 
necessary  to  give  here  a  few  of  the  other  reactions  without  entering  into 
details. 

Smith's  Reaction.  Pour  carefully  over  the  urine  some  tincture  of  iodine, 
whereby  a  green  ring  appears  between  the  two  liquids.  The  urine  may  also  be 
shaken  with  the  tincture  of  iodine  until  it  has  a  green  color. 

Ehrlich's  Test.  First  mix  the  urine  with  an  equal  volume  of  dilute  acetic 
acid  and  then  add  drop  by  drop  a  solution  of  sulphodiazobenzene.  The  acid 
mixture  becomes  dark  red  in  the  presence  of  bilirubin,  and  this  color  becomes 
bluish-violet  on  the  addition  of  glacial  acetic  acid.  The  sulphodiazobenzene  is  pre- 
pared by  mixing  1  pram  of  sulphanilic  acid,  15  c.  c.  of  hydrochloric  acid,  and  0.1  gram 
of  sodium  nitrite:  this  solution  is  diluted  to  1  liter  with  water.  This  test  is  not 
successful  and  positive  when  directly  applied  if  the  urine  is  rich  in  other  pigments. 

Medicinal  coloring-matters  produced  from  santonin,  rhubarb,  senna,  etc., 
may  give  an  abnormal  color  to  the  urine  and  maybe  mistaken  for  bile-pigments; 
or,  in  alkaline  urines,  perhaps  for  blood-coloring  matters.  If  hydrochloric  acid 
is  added  to  such  a  urine,  it  becomes  yellow  or  pale  yellow,  while  on  the  addi- 
tion of  an  excess  of  alkali  it  takes  on  a  more  or  less  beautiful  red  color. 

1  Deutech.  med.  Wochenschr.,  1902. 
'Deutsch.  Arch.  f.  klin.  Med..  78. 


8U0AR  IX   THE   rniNE.  561 


Sugar  in  Urine. 


The  occurrence  of  traces  of  dextrose  in  the  urine  of  perfectly  healthy 
persons  has  been,  as  above  stated  (page  521),  quite  positively  proved.  If 
BUgar  appears  in  the  urine  in  constant  and  especially  in  large  quantities,  ir 
must  lie  considered  as  an  abnormal  constituent.  In  a  previous  chapter 
several  of  the  principal  causes  of  glycosuria  in  man  and  animals  were 
mentioned,  and  the  reader  is  referred  to  Chapters  VIII  and  IX  for  the 
itial  facts  in  regard  to  the  appearance  of  sugar  in  the  urine. 

In  man  the  appearance  of  dextrose  in  the  urine  has  been  observed  under 
various  pathological  conditions,  such  as  lesions  of  the  brain  and  especially 
of  the  medulla  oblongata,  abnormal  circulation  in  the  abdomen,  diseases 
of  the  heart,  lungs,  and  liver,  cholera,  and  many  other  diseases.  The 
continued  presence  of  sugar  in  human  urine,  sometimes  in  very  consider- 
quantities,  occurs  in  diabetes  mellitus.  In  this  disease  there  may 
i  elimination  of  1  kilogram  or  even  more  of  dextrose  per  day.  In 
the  beginning  of  the  disease,  when  the  quantity  of  sugar  is  still  very  small, 
the  urine  often  does  not  appear  abnormal.  In  the  more  developed,  typical 
cases  the  quantity  of  urine  voided  increases  considerably,  to  3-6-10  liters 
per  day.  The  percentage  of  the  physiological  constituents  is  as  a  rule  very 
low,  while  their  absolute  daily  quantity  is  increased.  The  urine  is  pale, 
but  of  a  high  specific  gravity,  1.030-1.040  or  even  higher.  The  high  spe- 
cific gravity  depends  upon  the  quantity  of  sugar  present,  which  varies 
in  different  cases,  but  may  reach  10  per  cent.  The  urine  is  therefore 
characterized  in  typical  cases  of  diabetes  by  the  very  large  quantity  voided, 
by  the  pale  color  and  high  specific  gravity,  and  by  its  containing  sugar. 

That  the  urine  after  the  introduction  into  the  system  of  certain  medic- 
inal agents  or  poisonous  bodies  contains  reducing  substances,  conjugated 
glucuronic  acids,  which  may  be  mistaken  for  sugar,  has  already  been  men- 
tioned. 

The  properties  and  reactions  of  dextrose  have  been  considered  in  a  pre- 
vious chapter,  and  it  remains  but  to  mention  the  methods  of  detection  and 
quantitative  determination  of  dextrose  in  the  urine. 

The  detection  of  sugar  in  the  urine  is  ordinarily,  in  the  presence  of  not 
too  small  quantities,  a  very  simple  task.  The  presence  of  only  very  small 
quantities  may  make  its  detection  sometimes  very  difficult  and  laborious. 
A  urine  containing  proteid  must  first  have  the  proteid  removed  by  coagu- 
lation with  acetic  acid  and  heat  before  it  can  be  tested  for  sugar. 

The  tests  which  arc  most  frequently  employed  and  are  especially  recom- 
mended are  as  follows : 

Trommer's  Test.  In  a  typical  diabetic  urine  or  one  rich  in  sugar  this 
test  succeeds  well,  and  it  may  be  performed  in  the  manner  suggested  on 
page  94.  This  test  may  lead  to  very  great  mistakes  in  urines  poor  in  sugar, 
especially  when  the}-  have  at  the  same  time  normal  or  increased  amounts  of 


562  URINE. 

physiological  constituents,  and  therefore  it  cannot  be  recommended  to 
physicians  or  to  persons  inexperienced  in  such  work.  Normal  urine  con- 
tains reducing  substances,  such  as  uric  acid,  creatinine,  and  others,  and 
therefore  a  reduction  takes  place  in  all  urines  on  using  this  test.  A  separa- 
tion of  copper  suboxide  does  not  generally  occur,  but  still  if  one  varies  the 
proportion  of  the  alkali  to  the  copper  sulphate  and  "boils,  there  takes  place 
an  actual  separation  of  suboxide  in  normal  urines,  or  a  peculiar  yellowish- 
red  liquid  due  to  finely  divided  cuprous  hydrate.  This  occurs  especially 
on  the  addition  of  much  alkali  or  too  much  copper  sulphate,  and  by  careless 
manipulation  the  inexperienced  worker  may  therefore  sometimes  obtain 
apparently  positive  results  in  a  normal  urine.  On  the  other  hand,  as  the 
urine  contains  substances,  such  as  creatinine  and  ammonia  (from  the 
urea),  which  in  the  presence  of  only  a  little  sugar  may  keep  the  copper 
suboxide  in  solution,  the  investigator  may  easily  overlook  small  quantities 
of  sugar  that  may  be  present. 

Trommer's  test  may  of  course  be  made  positive  and  useful,  even  in  the 
presence  of  very  small  amounts  of  sugar,  by  using  the  modification  sug- 
gested by  Worm  Muller.  As  this  modification  is  rather  complicated 
and  requires  much  practice  and  exactness,  it  is  probably  rarely  employed 
by  the  busy  physician.     The  following  test  is  to  be  preferred. 

Almen's  bismuth  test,  which  recently  has  been  incorrectly  called  Nylan- 
der's  test,  is  performed  with  the  alkaline  bismuth  solution  prepared  as 
above  described  (page  94).  For  each  test  10  c.  c.  of  urine  is  taken  and 
treated  with  1  c.  c.  of  the  bismuth  solution  and  boiled  for  a  few  minutes. 
In  the  presence  of  sugar  the  urine  becomes  darker  yellow  or  yellowish 
brown.  Then  it  grows  darker,  cloudy,  dark  brown,  or  nearly  black,  and 
non-transparent.  After  a  longer  or  shorter  time  a  black  deposit  appears, 
the  supernatant  liquid  gradually  clears,  but  still  remains  colored.  In  the 
presence  of  only  very  little  sugar  the  test  does  not  become  black  or  dark 
brown,  but  simply  deeper  colored,  and  not  until  after  some  time  is  there 
seen  on  the  upper  layer  of  the  phosphate  precipitate  a  dark  or  black  layer 
(of  bismuth?).  In  the  presence  of  much  sugar  a  larger  amount  of  the 
reagent  may  be  used  without  disadvantage.  In  a  urine  poor  in  sugar  only 
1  c.  c.  of  the  reagent  for  every  10  c.  c.  of  the  urine  must  be  emploj^ed. 

This  test  shows  the  presence  of  0.5  p.  m.  sugar  in  the  urine.  The 
sources  of  error  which  interfere  in  Trommer's  test,  such  as  the  presence 
of  uric  acid  and  creatinine,  entirely  disappear  here.  The  bismuth  test  is, 
besides,  more  easily  performed,  and  it  is  therefore  to  be  recommended  to 
the  physician.  Small  quantities  of  proteid  do  not  interfere  with  this  test ; 
large  quantities  may  however  give  rise  to  an  error  by  forming  bismuth  sul- 
phide, and  therefore  it  is  better  to  remove  the  proteid  by  coagulation. 

In  using  this  method  it  must  not  be  overlooked  that  it  is,  like  Trom- 
mer's test,  a  reduction  test,  and  consequently  may  show,  besides  sugar, 
certain  other  reducing  substances.  Such  bodies  are  various  conjugated 
glucuronic  acids  which  may  appear  in  the  urine.  Positive  results  have 
been  obtained  with  the  bismuth  test  on  the  urine  after  the  use  of  severaL 
medicinal  agents,  such  as£rhubarb,  senna,  antinvrine,  kairin,  salol,  turpen- 
tineAand  others.  From  this  it  follows  that  we  shoukFnever'TDe  satisfied 
with  this  test  alone,  especially  when  the  reduction  is  not  very  great.  When 
this  test  gives  negative  results  the  urine  can  be  considered  from  a  clinical 
standpoint  as  free  from  sugar,  and  when  it  gives  positive  results  other  test-i 
must  be  applied.     Among  these  the  fermentation  test  is  of  special  value. 


SUGAR  IN  THE   URINE.  563 

Fermentation  Test.  On  using  this  tost,  the  process  must  vary  accord- 
ing as  the  bismuth  test  shows  small  or  large  quantities.  If  a  rather 
strong  reduction  is  obtained,  the  urine  may  be  treated  with  yeast  and  the 
presence  of  sugar  determined  by  the  generation  of  carbon  dioxide.  In 
this  case  the  acid  urine,  or  that  faintly  acidified  with  tartaric  acid,  is 
treated  with  yeast  which  has  previously  been  washed  by  decantation  with 
water.  Pour  this  urine  to  which  the  yeast  has  been  added  into  a  S<  HROT- 
tkk's  gas-burette,  or  glass  tube  with  the  open  end  ground,  close  with  the 
thumb,  and  open  under  the  surface  of  mercury  contained  in  a  dish.  As 
the  fermentation  proceeds,  the  carbon  dioxide  collects  in  the  upper  part  of 
the  tube,  while  a  corresponding  quantity  of  liquid  is  expelled  below.  As 
a  control  in  this  case  two  similar  tests  must  be  made,  one  with  normal 
urine  and  yeast  to  learn  the  quantity  of  gas  usually  developed,  and  the 
other  with  a  sugar  solution  and  yeast  to  determine  the  activity  of 
the  yeast. 

If,  on  the  contrary,  only  a  faint  reduction  with  the  bismuth  test  is 
found,  no  positive  conclusion  can  be  drawn  from  the  absence  of  any  carbon 
dioxide  or  the  appearance  of  a  very  insignificant  quantity.  The  urine 
absorbs  considerable  amounts  of  carbon  dioxide,  and  in  the  presence  of 
only  small  amounts  of  sugar  the  fermentation  test  as  above  per- 
formed may  lead  to  negative  or  inaccurate  results.  In  this  case  proceed 
in  the  following  way:  Treat  the  acid  urine,  or  the  urine  which  has  been 
faintly  acidified  with  tartaric  acid,  with  yeast  whose  activity  has  been 
tested  by  a  special  test  on  a  sugar  solution,  and  allow  it  to  stand  24-48 
hours  at  the  temperature  of  the  room,  or,  better,  at  a  little  higher  tem- 
perature. Then  test  again  with  the  bismuth  test,  and  if  the  reaction 
now  gives  negative  results,  then  sugar  was  previously  present.  But  if  the 
reaction  continues  to  give  positive  results,  then  it  shows,  if  the  yeast  is 
active,  the  presence  of  other  reducing,  unfermentable  substances.  There 
remains  of  course  the  possibility  that  the  urine  also  contains  some  sugar 
besides  these  bodies.  This  possibility  may  be  determined  by  the  follow- 
ing test: 
/"  Phenylhydrazine  Test.  According  to  v.  Jaksch  this  test  is  performed 
/  in  the  following  way:  Add  in  a  test-tube  containing  8-10  c.  c.  of  the  urine 
/  two  knife-points  of  phenylhydrazine  hydrochloride  and  three  knife-points 
of  sodium  acetate,  and  when  the  salts  do  not  dissolve  on  warming  add 
\  more  water.  The  test-tube  is  placed  in  boiling  water  and  warmed  on 
\  the  water-bath.  It  is  then  placed  in  a  beaker  of  cold  water.  If  the 
quantity  of  sugar  present  is  not  too  small,  a  yellow  crystalline  precipi- 
tate is  now  obtained.  If  the  precipitate  appears  amorphous,  there  are 
<  found,  on  looking  at  it  under  the  microscope,  yellow  needles  singly  and  in 
groups.  If  very  little  sugar  is  present,  pour  the  test  into  a  conical  glass 
and  examine  the  sediment.  In  this  case  at  least  a  few  phenylglucosazone 
crystals  are  found,  while  the  occurrence  of  larger  and  smaller  yellow  plates 
or  highly  refractive  brown  globules  does  not  show  the  presence  of  sugar. 
This  reaction  is  very  reliable,  and  by  it  the  presence  of  0.3  p.  m.  sugar  can 
be  detected  (Rosenfeld,  Geyer  1).  In  doubtful  cases  where  certainty 
is  desired,  prepare  the  crystals  from  a  large  quantity  of  urine,  dissolve  them 

1  Rosenfeld,  Deutsch.  med.  Wochenschr.,  1888;  Geyer,  cited  from  Roos,  Zeitschr. 
f.  physiol.  Chem.  15. 


564  URINE. 

on  the  filter  by  pouring  over  them  hot  alcohol,  treat  the  filtrate  with  water, 
and  boil  off  the  alcohol.  Still  better,  the  precipitate  is  dissolved,  accord- 
ing to  Neuberg,  in  some  pyridine  and  again  precipitated  as  crystals  by  the 
addition  of  benzene,  ligroin,  or  ether.  If  the  characteristic  yellow  crystal- 
line needles,  whose  melting-point  (204-205°  C.)  may  also  be  determined,  are 
now  obtained,  then  this  test  is  decisive  for  the  presence  of  sugar  It  must 
not  be  forgotten  that  lsevulose  gives  the  same  osazone  as  dextrose,  and 
that  a  further  investigation  is  necessary  in  certain  cases. 

The  following  modification  by  A.  Neumann  1  is  simple,  practical,  and 
at  the  same  time  sufficiently  delicate.  5  c.  c.  of  the  urine  are  treated  with 
2  c.  c.  of  acetic  acid  (30  per  cent)  saturated  with  sodium  acetate,  2  drops 
of  pure  phenylhydrazine  added  and  the  mixture  boiled  in  a  test-tube  until 
it  measures  3  c.  c.  After  quickly  cooling  warm  again  and  then  allow  it  to 
cool  slowly.  After  5-10  minutes  beautifully  formed  crystals  are  obtained 
even  in  the  presence  of  only  0.2  p.  m.  sugar. 

The  value  of  the  phenylhydrazine  test  has  been  considerably  debated, 
and  the  objection  has  been  made  that  glucuronic  acid  also  gives  a  similar 
precipitate.  A  confounding  with  glucuronic  acid  is,  according  to  Hirschl, 
not  to  be  apprehended  when  the  test  is  not  heated  in  the  water-bath  for  too 
short  a  time  (one  hour).  Kistermann  found  this  precaution  insufficient, 
and  Roos  states  that  the  phenylhydrazin  test  always  gives  a  positive 
result  with  human  urine,  which  coincides  with  E.  Holmgren  's  2  and  Ham- 
marsten's  experience.  This  test  only  shows  a  non-physiological  quan- 
tity of  sugar  when  a  rather  abundant  crystallization  is  obtained  from  a 
small  quantity  of  urine  (about  5  c.  c). 

Rubner's  test  is  performed  as  follows:  The  urine  is  precipitated  by 
an  excess  of  a  concentrated  lead-acetate  solution  and  the  filtrate  carefully 
treated  with  enough  ammonia  to  produce  a  flocculent  precipitate.  It  is 
then  heated  to  boiling,  when  the  precipitate  becomes  flesh-colored  or  pink 
in  the  presence  of  sugar. 

Polarization.  This  test  is  of  great  value,  especially  as  in  many  cases  it 
quickly  differentiates  between  dextrose  and  other  reducing,  lsevogyrate  sub- 
stances, such  as  the  conjugated  glucuronic  acids.  In  the  presence  of  only 
very  little  sugar  the  value  of  this  test  depends  on  the  delicacy  of  the  instru- 
ment and  the  dexterity  of  the  observer;  therefore  this  method  is  perhaps 
inferior  in  most  cases  to  the  bismuth  or  the  phenylhydrazine  test. 

If  small  quantities  of  sugar  are  to  be  isolated  from  the  urine,  precipitate 
the  urine  first  with  sugar  of  lead,  filter,  precipitate  the  filtrate  with  am- 
moniacal  basic  lead  acetate,  wash  this  precipitate  with  water,  decompose  it 
with  H2S  when  suspended  in  water,  concentrate  the  filtrate,  treat  it  with 
strong  alcohol  until  it  is  80  vol.  per  cent,  filter  when  necessary,  and  add 
an  alcoholic  caustic-alkali  solution.  Dissolve  the  precipitate  consisting  of 
saccharates  in  a  little  water,  precipitate  the  potash  by  an  excess  of  tartaric 
acid,  neutralize  the  filtrate  with  calcium  carbonate  in  the  cold,  and  filter. 
The  filtrate  may  be  used  for  testing  with  the  polariscope  as  well  as  for  the 
fermentation,  bismuth,  and  phenylhydrazine  tests.  The  presence  of  dex- 
trose may  be  detected  by  this  same  process  in  animal  fluids  or  tissues  from 

1  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl.  See  also  Margulies,  Berlin,  klin.  Wochen- 
schr.,  1900. 

2  Hirschl,  Zeitschr.  f.  physiol.  Chem.,  14;  Kistermann,  Deutsch.  Arch.  f.  klin.. 
Med.,  50;   Roos,  1.  c;   Holmgren,  Maly's  Jahresber.,  27 


SUGAR  IN   THE   URINE.  565 

which  the  proteidfl  have  been  removed  by  coagulation  or  by  the  addition 
of  alcohol. 

In  the  isolation  of  sugar  and  carbohydrates  from  the  urine  the  benzoic- 
acid  esters  of  the  same  may  be  prepared  according  to  Baumann's  method. 
The  urine  is  made  alkaline  with  caustic  soda  to  precipitate  the  earthy  phos- 
phates, the  filtrate  treated  with  10  c.  c.  of  benzoyl  chloride  and  120  c.  c.  of  10 
percent  caustic-soda  Bolutionfor  every  100  c. c. of  the  filtrate (Reinbold  x), 
and  shaken  until  the  odor  of  benzoyl  chloride  has  disappeared.  After 
standing  sufficiently  long  the  ester  is  collected,  finely  divided,  and  Baponified 
with  an  alcoholic  solution  of  sodium  ethylate  in  the  cold  according  to 
Baisch's  method,3  and  the  various  carbohydrates  separated  according  to 
his  suggestion. 

To  the  physician,  who  naturally  wants  simple  and  quick  methods,  the 
bismuth  test  is  especially  to  be  recommended.     If  this  test  gives  negative 
results,  the  urine  is  to  be  considered  as  free  from  sugar  in  a  clinical  s< 
If  it  gives  positive  results,  the  presence  of  sugar  must  be  controlled  by 
other  tests,  especially  by  the  fermentation  test. 

Other  tests  for  sugar,  as,  for  example,  the  reaction  with  orthonitrophenyl- 
propiolic  acid,  picric  acid,  diazobenzene-sulphonic  acid,  are  superfluous.  The 
reaction  with  tt-naphthol,  which  is  a  reaction  for  carbohydrates  in  general,  for 
glucuronic  acid  and  mucin,  may,  because  of  its  extreme  delicacy,  give  rise  to 
mistakes,  and  is  therefore  not  to  be  recommended  to  physicians.  Normal  urines 
give  this  test,  and  if  the  strongly  diluted  urine  gives  this  reaction  the  presence 
may  he  suspected  of  large  quantities  of  carbohydrates.  In  these  cases  more 
positive  results  are  obtained  by  using  other  tests.  This  test  requires  great 
cleanliness,  and  it  has  this  inconvenience,  that  it  is  very  difficult  to  get  sufficiently 
pure  sulphuric  acid.  Several  investigators,  such  as  v.  UdrANSKY,  Luther,  Roos 
and  Trbupel,8  have  investigated  this  test  in  regard  to  its  applicability  as  an 
approximate  test  for  carbohydrates  in  the  urine. 

Quantitative  Determination  of  Sugar  in  the  Urine.  The  urine  for  such 
an  estimation  must  first  be  tested  for  proteid,  and  if  any  be  present  it  must 
be  removed  by  coagulation  and  the  addition  of  acetic  acid,  care  being  taken 
not  to  increase  or  diminish  the  original  volume  of  urine.  The  quantity  of 
sugar  may  be  determined  by  titration  with  Feiiltxu's  or  Knapp's  solu- 
tion, by  fermentation,  by  polarization,  or  also  in  other  ways. 

The  titration  liquids  not  only  react  with  sugar,  but  also  with  certain 
other  reducing  substances,  and  on  this  account  the  titration  methods  give 
rather  high  results.  When  large  quantities  of  sugar  are  present,  as  in  typi- 
cal diabetic  urine,  which  generally  contains  a  lower  percentage  of  normal 
reducing  constituents,  this  is  indeed  of  little  account;  but  when  small  quan- 
tities of  sugar  are  present  in  an  otherwise  normal  urine,  the  mistake  nun-, 
on  the  contrary,  be  important,  as  the  reducing  power  of  normal  urine  may 
correspond  to  5  p.  m.  dextrose  (see  page  521).  In  such  cases  the  titra- 
tion procedure  must  be  employed  in  connection  with  the  fermentation 
method,  which  will  be  described  later.  It  is  to  be  remarked  that  in  typical 
diabetic  urines  with  considerable  quantities  of  sugar  the  titration  with 
Fehling's  solution  is  just  as  reliable  as  with  Knapp's  solution.  When 
the  urine,  on  the  contrary,  contains  only  little  sugar  with  normal  amounts 

1  Pfluger's  Arch.,  91. 

2  Zeitschr.  f.  physiol.  Chem.,  19. 

3  See  Roos  and  Treupel,  Zeitschr.  f.  physiol.  Chem.,  15  and  16. 


566  URINE. 

of  physiological  constituents,  then  the  titration  with  Fehling's  solution 
is  more  difficult,  in  certain  cases  .indeed  almost  impossible,  and  the  results 
become  very  uncertain.  In  such  cases  Knapp's  method  gives  good  results, 
according  to  Worm  Muller  and  his  pupils.1 

The  titration  with  Fehling's  solution  depends  on  the  power  of 
sugar  to  reduce  copper  oxide  in  alkaline  solutions.  For  this  there  was 
formerly  employed  a  solution  which  contained  a  mixture  of  copper  sulphate, 
Rochelle  salt,  and  sodium  or  potassium  hydrate  (Fehling's  solution);  but 
as  such  a  solution  readily  changes,  use  is  made  of  a  copper-sulphate  solu- 
tion and  an  alkaline  Rochelle-salt  solution  prepared  separately,  and  the 
two  solutions  mixed  in  equal  volumes  before  using. 

The  concentration  of  the  copper-sulphate  solution  is  such  that  10  c.  c.  of 
this  solution  is  reduced  by  0.05  gram  of  dextrose.  The  copper-sulphate 
solution  contains  34.65  grams  of  pure,  crystalled,  non-efflorescent  copper  sul- 
phate in  1  liter.  The  sulphate  is  crystallized  from  a  hot  saturated  solution 
by  cooling  and  stirring,  and  the  crystals  are  separated  from  the  mother- 
liquor  and  pressed  between  blotting-paper  until  dry.  The  Rochelle-salt 
solutionis  prepared  by  dissolving  173  grams  of  the  salt  in  350  c.c.  of  water, 
adding  600  c.  c.  of  a  caustic-soda  solution  of  a  specific  gravity  of  1.12,  and 
diluting  with  water  to  1  liter.  According  to  Worm  Muller,  these  three 
liquids — Rochelle-salt  solution,  caustic  soda,  and  water — should  be  sepa- 
rately boiled  before  mixing  together.  For  each  titration  mix  in  a  small 
flask  or  porcelain  dish  exactly  10  c.  c.  of  the  copper-sulphate  solution  and 
10  c.  c.  of  the  alkaline  Rochelle-salt  solution  and  add  30  c.  c.  of  water. 

The  urine,  freed  from  proteid,  is  diluted  with  water  before  the  titration, 
so  that  10  c.  c.  of  the  copper  solution  requires  between  5  and  10  c.  c.  of 
the  diluted  urine,  which  corresponds  to  between  1  per  cent  and  \  per  cent  of 
sugar.  A  urine  of  a  specific  gravity  of  1.030  may  be  diluted  five  times; 
one  more  concentrated,  ten  times.  The  urine  so  diluted  is  poured  into  a 
burette  and  allowed  to  flow  into  the  boiling  copper-sulphate  and  Rochelle- 
salt  solution  until  the  copper  oxide  is  completely  reduced.  This  has  taken 
place  when,  immediately  after  boiling,  the  blue  color  of  the  solution  disap- 
pears. It  is  very  difficult  and  requires  some  practice  to  exactly  determine 
this  point,  especially  when  the  copper  suboxide  settles  with  difficulty.  To 
determine  whether  the  color  has  disappeared,  allow  the  copper  suboxide 
to  settle  a  little  below  the  meniscus  formed  by  the  surface  of  the  liquid. 
If  this  layer  is  not  blue,  the  operation  is  repeated,  adding  0.1  c.  c.  less  of 
urine;  and  if,  after  the  copper  suboxide  has  settled,  the  liquid  has  a  blue 
color,  the  titration  may  be  considered  as  completed.  Because  of  the 
difficulty  in  obtaining  this  point  exactly  another  end-reaction  has  been 
suggested.  This  consists  in  filtering  immediately  after  boiling  a  small 
portion  of  the  titrated  mixture  through  a  small  filter  into  a  test-tube 
which  contains  a  little  acetic  acid  and  a  few  drops  of  potassium- 
ferrocyanide  solution  and  water.  The  smallest  quantity  of  copper  is 
shown  by  a  red  coloration.  If  the  operation  is  quickly  conducted  so  that 
no  oxidation  of  the  suboxide  into  oxide  takes  place,  this  end-reaction  is 
of  value  for  urines  which  are  rich  in  sugar  and  poor  in  urea  and  which  have 
been  strongly  diluted  with  water.  In  urines  poor  in  sugar  which  contain 
the  normal  amount  of  urea  and  which  have  not  been  considerably  diluted,  a 
considerable  quantity  of  ammonia  may  be  formed  from  the  urea  on  boil- 

1  Pfluger's  Arch.,  16  and  23;  Otto,  Journal  f.  prakt.  Chem.  (N.  F.),  26. 


ESTIMATION  OF  SUGAR.  567 

ing  the  alkaline  liquid.  This  ammonia  dissolves  the  suboxide  in  part, 
which  then  easily  passes  into  oxide,  and  besides  thia  the  dissolved  sub- 
oxide gives  a  red  color  with  potassium  ferrocyanide.     In  just  those  cases 

in  which  the  titration  is  most  difficult  this  end-reaction  is  the  Least  reliable 
Tract  ice  also  renders  it  unnecessary,  and  it  is  therefore  best  to  depend 
simply  upon   the  appearance  of  the  liquid. 

To  facilitate  the  settling  of  the  copper  suboxide  and  thereby  clearing 
the  liquid,  Munk  '  has  lately  suggested  the  addition  of  a  little  calcium- 
chloride  solution  ami  boiling  again.  A  precipitate  of  calcium  tartrate  i- 
produced  which  carries  down  the  suspended  copper  suboxide  with  it.  and 
the  color  of  the  liquid  can  then  be  better  seen.  This  artifice  succeeds  in 
many  cases,  but  unfortunately  there  are  urines  in  which  the  titration  with 
Fehling  's  solution  in  no  way  gives  exact  results.  In  those  cases  in  which 
only  small  quantities  of  sugar  exist  in  a  urine  rich  in  physiological  con- 
stituents it  is  best  to  dissolve  a  very  exactly  weighed  quantity  of  pure 
dextrose  or  dextrose-sodium  chloride  in  the  urine.  The  urine  can  now  be 
strongly  diluted  with  water  and  the  titration  becomes  successful.  The  dif- 
ference between  the  sugar  added  and  that  found  by  titration  give-  the 
reducing  power  of  the  original  urine  calculated  as  dextrose. 

The  necessary  conditions  for  the  success  of  the  titration  under  all  cir- 
cumstances are,  according  to  Soxhlet,2  the  following:  The  copper-sul- 
phate and  Rochelle-salt  solution  must,  as  above,  be  diluted  to  50  c.  c.  with 
water;  the  urine  should  contain  only  between  0.5  percent  and  1  per  cent  of 
su1  ar,  and  the  total  quantity  of  urine  required  for  the  reduction  must  be 
added  to  the  titration  liquid  at  once  and  boiled  with  it.  From  this  last 
condition  it  follows  that  the  titration  is  dependent  upon  minute  details, 
and  several  titrations  are  required  for  each  determination. 

It  is  best  to  give  here  an  example  of  the  titration.  The  proper  amount 
of  copper  sulphate  ami  Rochelle-salt  solution  and  water  (total  volume=50 
c.  c.)  is  heated  to  boiling  in  a  flask;  the  color  must  remain  blue.  The  urine 
diluted  five  times  is  now  added  to  the  boiling-hot  liquid,  1  c.  c.  at  a  time; 
after  each  addition  of  urine  boil  for  a  few  seconds  and  look  for  the  appear- 
ance <>f  the  end-reaction.  If  one  finds,  for  example,  that  3  c.  c.  is  too  little, 
but  that  4  c  c.  is  too  much  (the  liquid  becoming  yellowish),  then  the  urine 
has  not  been  sufficiently  diluted,  for  it  should  require  between  5  and  10  c.  c. 
of  the  urine  to  produce  the  complete  reduction.  The  urine  is  now  diluted 
ten  times,  and  it  should  require  between  6  and  S  c.  c.  for  a  total  reduction. 
Now  prepare  for  new  tests,  which  are  boiled  simultaneously  to  save  time, 
and  add  at  one  time  respectively  6,  6£,  7,  and  7£  c.  c.  of  urine.  If  it  is 
found  that  between  6£  and  7  c.  c.  are  necessary  to  produce  the  end-reac- 
tion, then  make  four  other  tests,  to  which  add  respectively  6.6,  6.7,  6.8, 
and  6.9  c.  c.  of  urine.  If  in  this  case  the  liquid  is  still  somewhat  bluish 
With  6.7  c.  c.  and  completely  decolorized  with  6.8  c.  c.  the  average  fig-, 
ure  6.75  c.  c.  is  considered  as  correct. 

The  calculation  is  simple.     The  6.75  c.  c.  used   contains  0.05  gram  of 

sugar,  and  the  percentage  of  sugar  in  the  dilute  urine  is  therefore  (6.75:0.05  = 
5 

100:z)  =  — ^  =  0.74.     But  as  the  urine  was  diluted  with  ten  times  its  vol- 

5x  10 

ume  of  water,  the  undiluted  mine  contained  ' =-  =  7.4  per  cent.     The 

o.i  5 


1  Virchow'a  Arch..  105.  »  Journal  f.  prakt.  Chem.  (X.  F.),  21. 


56S  URINE. 

general  formula  on  using  10  c.  c.  of  copper-sulphate  solution  is  therefore 

5X  n 

— j— ,  in  which  n  represents  the  number  of  times  the  urine  has  been  diluted 

and  k  the  number  of  c.  c.  of  the  diluted  urine  employed  for  the  titration. 

The  titration  according  to  Knapp  depends  on  the  fact  that  mercuric 
cyanide  in  alkaline  solution  is  reduced  to  metallic  mercury  by  dextrose.. 
Ihe  titration  liquid  should  contain  10  grams  of  chemically  pure  dry 
mercuric  cyanide  and  100  c.  c.  of  caustic-soda  solution  of  a  specific  gravity 
of  1.145  per  liter.  When  the  titration  is  performed  as  described  below  (ac- 
cording to  Worm  Muller  and  Otto),  20  c.  c.  of  this  solution  should  cor- 
respond to  exactly  0.05  gram  of  dextrose.  If  the  process  is  carried  out  in. 
other  ways,  the  value  of  the  solution  is  different. 

In  this  titration  also  the  quantity  of  sugar  in  the  urine  should  be  between 
^  and  1  per  cent  and  the  extent  of  dilution  necessary  be  determined  by 
a  preliminary  test.  To  determine  the  end-reaction  as  described  below,, 
the  test  for  the  excess  of  mercuy  is  made  with  sulphuretted  Irydrogen. 

In  performing  the  titration  allow  20  c.  c.  of  Knapp 's  solution  to  flow 
into  a  flask  and  dilute  with  SO  c.  c.  of  water,  or  when  the  urine  contains 
less  than  0.5  per  cent  of  sugar,  use  only  40-60  c.  c.  After  this  heat  to 
boiling  and  allow  the  dilute  urine  to  flow  gradually  into  the  hot  solution, 
at  first  2  c.  c,  then  1  c.  c,  then  0.5  c.  c,  then  0.2  c.  c,  and  lastly  0.1  c.  c. 
After  each  addition  let  it  boil  |  minute.  When  the  end-reaction  is. 
approaching,  the  liquid  begins  to  clarify  and  the  mercury  separates  with 
the  phosphates.  The  end-reaction  is  determined  by  taking  a  drop  of  the 
upper  layer  of  the  liquid  into  a  capillary  tube  and  then  blowing  it  out 
on  pure  white  filter-paper.  The  moist  spot  is  first  held  over  a  bottle 
containing  fuming  hydrochloric  acid  and  then  over  strong  sulphuretted 
hydrogen.  The  presence  of  a  minimum  quantity  of  mercury  salt  in  the 
liquid  is  shown  by  the  spot  becoming  yellowish,  which  is  best  seen  when  it 
is  compared  with  a  second  spot  that  has  not  been  exposed  to  the  gas. 
The  end-reaction  is  still  clearer  when  a  small  part  of  the  liquid  is  filtered, 
acidified  with  acetic  acid,  and  tested  with  sulphuretted  hydrogen  (Otto  x)~ 
The  calculations  are  just  as  simple  as  for  the  previous  method. 

This  titration,  unlike  the  previous  one,  may  be  performed  equally  well 
in  daylight  and  in  artificial  light.  Knapp 's  method  has  the  following 
advantages  over  Fehling  's  method :  It  is  applicable  even  when  the  quan- 
tity of  sugar  in  the  urine  is  very  small  and  that  of  the  other  urinary 
constituents  is  normal.  It  is  more  easily  performed,  and  the  titration 
liquids  may  be  kept  without  decomposing  for  a  long  time  (Worm  Muller 
and  his  pupils  2) .  The  views  of  different  investigators  on  the  value  of  this 
titration  method  are  somewhat  contradictory. 

Besides  the  above-described  titration  methods  there  are  various  others. 
Thus  Pavy  titrates  with  an  ammoniacal  copper  solution.  K.  B.  Lehmann 
uses  an  excess  of  copper  salt  and  retitrates  with  potassium  iodide  and 
hyposulphite.  The  sugar  can  also  be  determined  according  to  Allihn, 
and  especially  according  to  Pfluger's  modification  of  this  method.3 

1  Journal  f .  prakt.  Chem. ,  20. 

2  Pfluger's  Arch.,  16  and  23. 

3  Lehmann,  Arch.  f.  Hygiene,  30;  Pfliiger,  Pfliiger's  Arch.,  06.  In  regard  to 
Pavy's  and  other  methods,  see  Huppert-Neubauer,  Ham- Analyse,  10.  Aufl. 


ESTIMATION   OF  SUGAR.  569 

Estimation  of  the  Quantity  of  Sugar  by  Fermentation.  This 
may  be  done  in  various  ways;  the  simplest  method,  and  one  at  the  same 
time  sufficiently  exact  for  ordinary  cases,  is  that  of  Roberts.  This  con- 
sists in  determining  the  specific  gravity  of  the  urine  before  and  after  fer- 
mentation. In  the  fermentation  of  sugar,  carbon  dioxide  and  alcohol  are 
formed  as  chief  products  and  the  specific  gravity  is  lowered,  partly  on 
account  of  the  disappearance  of  the  sugar  and  partly  on  account  of  the 
production  of  alcohol.  Roberts  found  that  a  decrease  of  0.001  in  the 
specific  gravity  corresponded  to  0.23  per  cent  sugar,  and  this  has  been  sub- 
stantiated since  by  several  other  investigators  (WORM  Muller  and  others). 
If  the  urine,  for  example,  has  a  specific  gravity  of  1.030  before  fermentation 
and  1.008  after,  then  the  quantity  of  sugar  contained  therein  was  22X0.23 
=  5.06  per  cent. 

In  performing  this  test  the  specific  gravity  must  be  taken  at  the  same 
temperature  before  and  after  the  fermentation.  The  urine  must  be  faintly 
acid,  and  when  necessary  it  should  be  acidified  with  a  little  tartaric-acid 
solution.  The  activity  of  the  yeast  must,  when  necessary,  be  controlled 
by  a  special  test.  Place  200  c.  c.  of  the  urine  in  a  400  c.  c.  flask,  add 
a  piece  of  compressed  yeast  the  size  of  a  pea,  and  subdivide  the  yeast 
through  the  liquid  by  shaking;  close  the  flask  with  a  stopper  provided  with 
a  finely-drawn-out  glass  tube,  and  allow  the  test  to  stand  at  the  temperature 
of  the  room  or,  still  better,  at  20-25°  C.  After  24—48  hours  the  fermenta- 
tion is  ordinarily  ended,  but  this  must  be  verified  by  the  bismuth  test. 
After  complete  fermentation  filter  through  a  dry  filter,  bring  the  filtrate  to 
the  proper  temperature,  and  determine  the  specific  gravity. 

If  the  specific  gravity  be  determined  with  a  good  pyenometer  supplied 
with  a  thermometer  and  an  expansion-tube,  this  method,  when  the  quan- 
tity of  sugar  is  not  less  than  4-5  p.  m.,  gives,  according  to  Worm  Miller, 
very  exact  results,  but  this  has  been  disputed  by  Budde.1  For  the  physi- 
cian the  method  in  this  form  is  not  quite  serviceable.  Even  when  the 
specific  gravity  is  determined  by  a  delicate  urinometer  which  can  give  the 
density  to  the  fourth  decimal,  quite  exact  results  are  not  obtained,  because 
of  the  ordinary  errors  of  the  method  (Budde);  but  the  errors  are  usually 
smaller  than  those  which  occur  in  titrations  made  by  unpractised  hands. 

When  the  quantity  of  sugar  is  less  than  5  p.  m.  these  methods  cannot 
be  used.  Such  small  amounts  camiot,  as  already  mentioned,  be  determined 
by  titration  directly,  because  the  reducing  power  of  normal  urine  corre- 
sponds to  4-5  p.  m.  of  sugar.  In  such  cases,  according  to  Worm  Muller, 
it  is  better  first  to  determine  the  reduction  power  of  the  urine  by  titration 
with  Knapp's  solution,  then  ferment  tne  urine  with  the  addition  of  yeast 
and  titrate  again  with  Knapp's  solution.  The  difference  found  between 
the  two  titrations  calculated  as  sugar  gives  the  true  quantity  of  the  latter. 

The  determination  of  the  sugar  by  fermentation  can  be  so  per- 
formed that  the  loss  in  weight  due  to  the  C02  can  be  estimated  or  the 
volume  of  the  gas  measured.  For  this  last  purpose  Lohnstf.in  2  has  con- 
structed a  special  fermentation  saccharometer  which  is  claimed  to  be 
trustworthy  and  practical. 

1  Roberts,  Edinburgh  Med.  Journ.,  1861,  and  The  Lancet,  1.  1802;  Worm  Muller, 
Pfluger's  Arch.,  33  and  3";  Budde,  ibid.,  40,  and  Zeitschr.  f.  physiol.  Chem.,  13  See 
also  Huppert-Xeubauer,  10.  Aufl.,  and  Lohnstein,  Pfluger's  Arch.,  62. 

2  Berlin,  klin.  Wochenschr.,  35,  and  Allg.  med.  Central. -Ztg.,  1S99. 


570  URINE. 

Estimation  of  Sugar  by  Polarization.  In  this  method  the  urine 
must  be  clear,  not  too  deeply  colored,  and,  above  all,  must  not  contain 
any  other  optically  active  substances  besides  dextrose.  The  urine  may 
contain  several  lsevorotatory  substances  such  as  proteids,  /?-oxybutyric 
acid,  conjugated  glucuronic  acids,  the  so-called  Leo's  sugar,  and  less  often 
cystin,  all  of  which  are  unfermentable.  The  proteid  is  removed  by  coagu- 
lation and  the  others  are  detected  by  the  polariscope  after  complete  fer- 
mentation. The  fermentable  lsevulose  is  detected  in  a  special  manner 
(see  below),  and  the  dextrorotatory  milk-sugar  differs  from  dextrose  in  its 
not  fermenting  readily.  By  using  a  delicate  instrument  and  with  suffi- 
cient practice,  very  exact  results  can  be  obtained  by  this  method.  The 
value  of  this  procedure  consists  in  the  rapidity  with  which  the  determina- 
tion can  be  made.  In  using  instruments  specially  constructed  for  clinical 
purposes  the  accuracy  is  less  than  with  the  less  expensive  fermentation 
test.  Under  such  circumstances,  and  as  the  estimation  by  means  of  polari- 
zation can  be  performed  with  exactitude  only  by  specially  trained  chemists, 
it  is  hardly  necessary  to  give  this  method  in  detail,  and  the  reader  is  referred 
to  handbooks  for  instructions  in  the  use  of  the  apparatus. 

Laevuloss.  Lsevogyrate  urines  containing  sugar  have  been  observed  by 
Ventzke,  Zimmer  and  Czapek,  Seegen,  and  others.  The  nature  of  the 
substance  causing  this  action  is  difficult  to  describe  exactly,  but  there  is 
hardly  any  doubt  that  the  urine,  at  least  in  certain  cases,  as  in  those  observed 
by  Seegen,  contains  lsevulose.  May  has  also  recently  published  a  case  in 
which  to  all  appearances  lsevulose  was  present.  Undoubted  cases  of  lsevu- 
losuria  have  been  observed  and  studied  in  the  last  few  years  by  Rosin  and 
Laband,  Spath  and  Weil.1 

Lsevulose  is  detected  as  follows:  The  urine  is  lsevorotatory,  and  the 
lsevorotatory  substance  ferments  with  yeast.  The  urine  gives  the  ordinary 
reduction  tests  and  phenylglucosazone.  It  gives  Seliwanoff's  reaction 
on  boiling  with  resorcin  and  hydrochloric  acid,  and  with  methylphenylhy- 
drazine  it  gives  the  characteristic  lsevulosemethylphenylosazone  (see  page  96). 

Laiose  is  a  substance  named  by  Htjppert  and  found  by  Leo  2  in  diabetic  urines 
in  certain  cases,  and  which  he  considers  as  a  sugar.  It  is  lsevogyrate,  amorphous, 
and  does  not  taste  sweet,  but  rather  sharp  and  salt}r.  Laiose  has  a  reducing 
action  on  metallic  oxides,  does  not  ferment,  and  gives  a  non-crystalline,  yellowish- 
brown,  oil  with  phenylhydrazine.  There  is  no  positive  proof  as  3ret  that  this 
substance  is  a  sugar. 

Lactose.  The  appearance  of  lactose  in  the  urine  of  pregnant  women 
•was  first  shown  by  the  observations  of  De  Sinety  and  F.  IIofmeistkk, 
anil  this  has  been  substantiated  by  other  investigators.3  After  the  inges- 
tion of  large  quantities  of  milk-sugar  some  lactose  may  be  found  in  the 
urine  (see  Chapter  IX  on  absorption).  The  passage  of  lactose  into  the  urine 
is  called  lactosuria. 

The  positive  detection  of  this  sugar  in  the  urine  is  difficult,  be- 
cause it  is,  like  dextrose,  dextrogyrate  and  also  gives  the  usual  reduction 

1  Rosin  and  Laband,  Zeitschr.  f.  klin.  Med.,  47;  Spilth  and  Woil,  Centralbl.  f.  d. 
med.  Wiss.,  1903.     See  also  Huppert-Xeubauer,  10.  Aufl.,  125. 

2Virchow's  Arch.,  107. 

s  Hofmeister,  Zeitschr  f.  physiol.  Chem.,  1,  which  also  contains  the  pertinent 
literature.     See  also  Lemaire,  ibid.,  21- 


PENTOSES  IN   THE   URINE.  571 

tests.  If  urine  contains  a  dextrogyrate,  non-fermentable  sugar  which 
reduces  bismuth  solutions,  then  it  is  very  probable  that  it  contains  lac- 
tose. It  must  be  remarked  that  the  fermentation  test  for  lactose  is, 
according  t<>  the  experience  of  LuSK  and  Yorr,1  best  performed  by  using 
pure  cultivated  yeast  (saccharomyces  apiculatus).  This  yeast  only  fer- 
ments the  dextrose,  while  it  does  not  decompose  the  milk-sugar.  If.  accord- 
ing to  VoiT,  RuBNEB  's  test  is  performed  without  heating  to  boiling  but  only 
to  so0  C.,  the  color  becomes  yellow  or  brown  in  the  presence  of  lactose, 
instead  of  red.  The  most  positive  means  for  the  detection  of  this  sugar  is 
to  isolate  the  sugar  from  the  urine.  This  may  be  done  by  the  following 
me'liod,  suggested  by  F.  Hofmeister: 

Precipitate  the  urine  with  sugar  of  lead,  filter,  wash  with  water,  unite  the 
filtrate  and  wash-water,  and  precipitate  with  ammonia.  The  liquid  filtered 
from  the  precipitate  is  again  precipitated  by  sugar  of  lead  and  ammonia  until  the 
last  filtrate  is  optically  inactive.  The  several  precipitates  with  the  exception 
of  the  first,  which  contains  no  sugar,  are  united  and  washed  with  water. 
This  precipitate  is  decomposed  in  the  cold  with  sulphuretted  hydrogen  and 
filtered.  The  excess  of  sulphuretted  hydrogen  is  driven  off  by  a  current  of  air; 
the  acids  set  free  are  removed  by  shaking  with  silver  oxide.  Now  filter,  remove 
by  sulphuretted  hydrogen  the  soluble  silver,  treat  with  barium  carbonate  to  unite 
with  any  free  acetic  acid  present,  and  concentrate.  Before  the  evaporated  residue 
becomes  syrupy  it  is  treated  with  90  per  cent  alcohol  until  a  flocculent  precipi- 
tate is  formed  which  settles  quickly.  The  filtrate  from  this  when  placed  in  a 
desiccator  deposits  crystals  of  lactose,  which  are  purified  by  recrystallization, 
decolorizing  with  animal  charcoal  and  boiling  with  60-70  per  cent  alcohol. 

Pentoses.  Salkowski  and  Jastrowitz  first  found  in  the  urine  of  per- 
sons addicted  to  the  morphine  habit  a  variety  of  sugar  which  was  a  pen- 
tose and  yielded  an  osazone  which  melted  at  159°  C.  This  and  several 
other  cases  of  pentosuria  have  been  observed,  and  according  to  Kulz  and 
Vogel  small  amounts  of  pentose  also  occur  in  the  urine  of  diabetics  as 
also  in  the  urine  of  dogs  with  pancreatic  or  phlorhizin  diabetes.2 

The  pentose  isolated  by  Xeuberg  from  the  urine  in  chronic  pento- 
suria was  i-arabinose.  In  alimentary  pentosuria  the  1-arabinose  of  the 
plant  food  may  be  found  in  the  urine. 

A  urine  containing  pentose  reduces  bismuth  as  well  as  copper  solutions, 
although  the  reduction  is  not  as  rapid  but  appears  gradually.  If  only 
pentose  is  present,  the  urine  does  not  ferment,  but  in  the  presence  of  dex- 
trose small  amounts  of  pentose  may  also  undergo  fermentation.  The 
preparation  of  the  osazone  serves  in  the  detection  of  pentoses ;  this  com- 
pound when  pure  melts  at  166-168°  C,  but  when  obtained  from  the  urine 
has  a  melting-point  of  156-160°  C.  The  phloroglucin  or  orcin  tests  can  also 
be  employed  (see  page  90).  Of  these  the  last  is  most  preferable,  especially 
as  it  excludes  a  confusion  with  the  conjugated  glucuronic  acids. 

1  Carl  Yoit,  Ueber  die  Glycogenbildung  nach  Aufnahme  verschiedener  Zuckeraten, 
Zeitschr.  f.  Biologie,  28. 

2  In  regard  to  the  literature,  see  foot-note  1,  page  89.  See  also  Blumenthal, 
"Die  Pentosurie,"  Deutsche  Klinik,  1902. 


572  URINE. 

The  orcin  test  can  be  performed  as  follows:  5  c.  c.  of  the  urine  is  mixed 
with  an  equal  volume  of  HC1  sp.  gr.  1.19  and  a  small  amount  of  orcin  added 
and  heated  to  boiling.  As  soon  as  a  greenish  cloudiness  appears,  cool 
the  mixture  off  and  shake  carefully  with  amyl  alcohol.  The  amyl-alcohol 
solution  is  used  in  the  spectroscopic  examination.  The  precipitation  of  a 
bluish-green  pigment  is  in  itself  significant. 

Bial  *  uses  as  reagent  30  per  cent  hydrochloric  acid  which  contains 
1  gram  of  orcin  and  25  drops  of  a  ferric-chloride  solution  (62.9  per  cent  of 
the  crystalline  salt)  in  500  c.  c.  of  the  acid.  4^5  c.  c.  of  the  reagent  is 
heated  to  boiling  and  then  a  few  drops  (not  more  than  1  c.  c.)  of  the  urine 
is  added  to  the  hot  but  not  boiling  liquid.  In  the  presence  of  pentose 
the  liquid  turns  a  beautiful  green.  Normal  or  diabetic  urines  do  not  give 
this  reaction,  neither  do  the  conjugated  glucuronic  acids. 

Lepine  and  Bonlud  2  have  shown  the  presence  of  maltose  in  cases  of 
diabetes.  After  boiling  with  hydrochloric  acid  the  specific  rotation  dimin- 
ishes, while  the  reducing  power  increases  in  such  urines. 

Conjugated  Glucuronic  Acids.  Certain  conjugated  glucuronic  acids  such 
as  menthol-  and  turpentine-glucuronic  acid  may  spontaneously  decompose  in 
the  urine  and  in  this  case  they  may  readily  lead  to  a  confusion  with  pentoses. 
The  urine  should  be  always  as  fresh  as  possible  for  these  examinations. 

A  confusion  of  the  glucuronic  acids  which  have  a  reducing  power  on 
copper  or  bismuth  solutions  with  dextrose  and  lsevulose  can  be  prevented 
by  the  fermentation  test.  The  optical  behavior  also  serves  as  a  difference, 
as  the  conjugated  glucuronic  acids  are  lsevogyrate.  On  boiling  with  an 
acid  dextrorotatory  glucuronic  acid  is  produced  and  the  lsevorotation  is 
changed  to  dextrorotation. 

The  conjugated  glucuronic  acids,  like  the  pentoses,  give  the  phloro- 
glucin-hydro chloric-acid  test.  On  the  contrary  they  do  not  give  the  orcin 
test  directly,  but  only  after  cleavage  with  the  setting  free  of  glucuronic 
acid.  On  using  Bial's  reagent  no  mistaking  for  pentoses  occurs.  The  pen- 
toses may  also  be  isolated  and  identified  by  their  osazones.  The  occurrence 
of  conjugated  glucuronic  acid  in  the  urine  is  shown  when  the  urine  does 
not  give  the  orcin-hydrochloric-acid  reaction  directly,  but  only  after  boiling 
with  the  acid.  A  further  proof  is  that  suggested  by  v.  Alfthan.3  500  c.  c. 
of  the  urine  is  benzoylated  and  the  ester  obtained  saponified  with  sodium 
ethylate.  The  free  and  conjugated  glucuronic  acid  is  thus  obtained  as 
sodium  compounds,  insoluble  in  alcohol,  while  the  pentoses,  if  present, 
remain  in  the  alcoholic  filtrate. 

The  surest  method  is  that  suggested  by  Mayer  and  Neuberg  *  which 
consists  in  precipitating  the  urine  with  basic  lead  acetate,  decomposing 
the  precipitate  with  H28,  boiling  with  dilute  sulphuric  acid  in  order  to 

1  Deutsch.  mcd.  Wochenschr.,  1903. 
2Compt.  rend.,  132. 

3  Arch   f.  exp.  Path.  u.  Pharm.,  47. 

4  Zeitschr.  f.  physiol.  Chem.,  29. 


ACETONE  BODIES  IN   THE   URINE.  573 

split  the  conjugated  acid  and  then  after  neutralizing  with  soda  prepare 
the  characteristic  bromphenylhydrazine  compound  of  glucuronic  acid  (see 
page  100)  with  p-bromphenylhydrazine  hydrochloride  and  sodium  acetate. 

Inositc  occurs  in  the  urine  in  albuminuria  and  in  diabetes  mellitus,  but 

only  rarely  and  in  small  quantities.  Inositc  is  also  found  in  the  urine 
after  the  excessive  drinking  of  water.  According  to  HoppE-SeylER  ' 
traces  of  inositc  occur  in  all  normal  urines. 

In  detecting  inositc  the  proteid  is  first  removed  from  the  urine.  Then  con- 
centrate the  urine  on  the  water-bath  to  i  of  its  original  volume  and  precipitate 
with  sugar  of  lead.  The  filtrate  is  warmed  and  treated  with  basic  lead  acetate  as 
long  as  a  precipitate  is  formed.  The  precipitate  formed  after  twenty-four  hours 
is  washed  with  water,  suspended  in  water,  and  decomposed  with  sulphuretted 
hydrogen.  A  little  uric  acid  may  separate  from  the  filtrate  after  a  short  time. 
The  liquid  is  filtered,  concentrated  to  a  syrupy  consistency,  and  treated  while 
boiling  with  3-4  vols,  alcohol.  The  precipitate  is  quickly  separated.  After 
the  addition  of  ether  to  the  cooled  filtrate,  crystals  separate  after  a  time,  and 
these  are  purified  by  decolorization  and  recrystallization.  With  these  crystals 
perform  the  tests  mentioned  on  page  387. 

Acetone  Bodies  (acetone,  acetoacetic  acid,  /?-oxybutyric  acid).  These 
bodies,  whose  occurrence  in  the  urine  and  formation  in  the  organism  have 
been  the  subject  of  numerous  investigations,  occur  in  the  urine  espe- 
cially in  diabetes  mellitus,  but  also  in  many  other  diseases.2  According  to 
v.  Jaksch  and  others  acetone  is  a  normal  urinary  constituent,  though  it 
may  occur  only  in  very  small  amounts  (0.01  gram  in  twenty-four  hours). 

In  regard  to  the  origin  of  these  bodies  it  was  previously  considered  that 
they  were  produced  by  an  increased  destruction  of  proteid.  One  of  the 
various  reasons  for  this  was  the  increase  in  the  elimination  of  acetone  and 
acetoacteic  acid  during  inanition  (v.  Jaksch,  Fr.  Muller  3).  This  stands 
also  in  good  accord  with  the  observations  that  a  considerable  increase  in 
f  the  quantity  of  acetone  and  acetoacetic  acid  eliminated  is  observed  in 
such  diseases  as  fevers,  diabetes,  digestive  disturbances,  mental  diseases 
with  abstinence  and  cachexia,  where  the  body  proteid  is  largely  destroyed. 
The  formation  of  acetone  bodies  from  proteid  is  also  indicated  by  the  fact 
that  acetone  has  been  obtained  as  an  oxidation  product  from  gelatine  and 
proteid  (Blumenthal  and  Xeuberg,  Orgler  *).  On  the  other  hand,  the 
facts,  as  shown  by  Weixtraud   and   Palm  a,  that  no   parallelism   exists 

1  Handbuch  d.  physiol.  u.  pathol.  chem.  Analyse,  6.  Aufl.,  196. 

2  In  regard  to  the  extensive  literature  on  acetone  bodies  the  reader  is  referred  to 
Huppert-Xeubauer,  Ham-Analyse,  10.  Aufl.,  and  v.  Xoorden's  Lehrb.  d.  Pathol,  des 
Stoffwechsels.     Berlin,  1893. 

3  v.  Jaksch,  Ueber  Acetonurie  und  Diaceturie.  Berlin,  1885;  Fr.  Muller,  Bericht 
uber  die  Ergebnisse  des  an  Cetti  ausgefiihrten  Hungerversuches.  Berlin,  klin.  Wochen- 
schr.,  18S7. 

4  Blumenthal  and  Xeuberg,  Deutsch.  med.  Wochenschr. ,  1901;  Orgler,  Hofmeis- 
ter's  Beitriige,  1. 


574  URINE. 

between  the  acetone  and  nitrogen  excretion  in  diabetics  as  claimed  espe- 
cially by  Wright,  and  that  in  man  no  certain  relationship  exists  between 
these  two  values,  show  that  no  such  origin  of  acetone  bodies  exists.  In 
man  the  excretion  of  acetone  does  not  increase  with  the  rise  in  the  quan- 
tity of  proteid  and  an  increase  in  the  latter  above  the  average  causes  a 
diminution  in  the  elimination  of  acetone  (Rosenfeld,  Hirschfeld,  Fr. 
Voit  1).  At  the  present  time  the  tendency  is  more  and  more  to  the  view 
that  the  acetone  bodies  do  not  originate  from  the  proteids  but  from  the 
fats;  if  they  are  not  the  only  source  they  are  at  least  the  most  important. 

The  carbohydrates  have  a  strong  influence  on  the  elimination  of  ace- 
tone, namely,  the  exclusion  of  carbohydrates  from  the  food,  or  the  dimi- 
nution in  their  amount,  causes  an  increased  elimination,  while  abundance 
of  carbohydrates  decreases  the  quantity  considerably,  or  even  causes  a  dis- 
appearance. The  increased  excretion  of  acetone  with  carbohydrate  star- 
vation occurs  also  in  healthy  individuals  with  a  fat-rich  diet,  or  on  the 
supply  of  sufficient  calories  in  other  ways  (alimentary  acetonuria) ;  and 
if  the  formation  of  acetone  from  proteids  is  not  accepted  it  must  be  ad- 
mitted as  regards  fats.  As  proof  for  this  there  are  certain  cases  of  diabetes 
with  strong  elimination  of  acetone  bodies  (/?-oxybutyric  acid)  where  the 
quantity  of  proteid  transformed  was  too  small  to  account  for  the  acetone 
bodies  (Magnus-Levy).  Certain  investigators  (Geelmuyden,  Schwarz,. 
Waldvogel  2)  have  also  observed  an  increase  in  the  acetonuria  on  par- 
taking of  fatty  food. 

There  is  no  doubt  that  the  fats  bear  a  certain  relationship  to  the  ace- 
tone bodies,  and  that  they  are  probably  in  part  the  source  of  the  same. 
It  has  not  been  proven,  on  the  contrary,  that  the  fats  are  the  only  or  the 
most  important  source  of  the  acetone  bodies.  Still  it  is  certain  that  in 
man,  insufficient  introduction  or  utilization  of  carbohydrates  may  lead  to 
a  large  or  small  acetone  excretion,  and  that  these  conditions  exist  in  dia- 
betes as  well  as  in  starvation,  and  also  in  the  above-mentioned  diseased 
conditions. 

In  drawing  conclusions  as  to  the  origin  of  the  acetone  bodies  it  must  not 
be  forgotten  that  the  conditions  are  distinctly  different  in  man  than  in  car- 
nivora  (Geelmuyden,  Fr.  Voit).  In  dogs  the  elimination  of  acetone 
bodies  is  not  increased  in  starvation,  but  is  reduced;  it  is  increased  with 
large  amounts  of  meat,  runs  parallel  with  the  nitrogen  excretion,  and  is 
not  diminished  by  carbohydrates  (Fr.  Voit). 

1  Hirschfeld,  Zeitschr.  f.  klin.  Med.,  28;  Geelmuyden,  see  Maly's  Jahresber.,  26, 
and  Zeitschr.  f.  physiol.  Chem.,  23  and  26;  Weintraud,  Arch.  f.  exp.  Path.  u.  Pharm.,. 
34;  Palma,  Zeitschr.  f.  Heilkunde,  15;  Wright,  Maly's  Jahresber.,  21;  Rosenfeld, 
Centralbl.  f.  innere  Med.,  16;  Voit,  Deutsch.  Arch.  f.  klin  Med.,  66. 

2  Magnus-Levy,  Arch.  f.  exp.  Path.  u.  Pharm.,  42;  Geelmuyden,  1.  c,  and  Norsk. 
Magazin  for  Laegevidenskaben,  1900;  Schwarz,  Deutsch.  Arch.  f.  klin.  Med.,  1903; 
"Waldvogel,  Centralbl.  f.  innere  Med.,  20. 


ACETONE.  575 

.CHS 
Acetone,    03ILO,    dimcthylketone==CO<T         ,  occurs,  as  above  stated, 

Vh, 

in  very  small  amounts  in  normal  urine.  In  diabetes  it  may  give  an  odor 
similar  to  apples  or  fruit  to  the  urine  as  well  as  to  the  expired  air. 

Irrespective  of  the  alimentary  acetonuria  derived  from  the  food,  there 
occurs  an  increased  elimination  of  acetone,  as  above  stated,  in  many  dis- 
eases, as  also  after  nervous  lesions,  certain  intoxications,  and  after  admin- 
istration of  phlorhizin  or  extirpation  of  the  pancreas  (v.  Mering  and  Min- 
kowski, AZEMAB  l). 

Acetone  is  a  thin  -water-clear  liquid  boiling  at  56.3°  C.  and  possessing  a 
pleasant  odor  of  fruit.  It  is  lighter  than  water,  with  which  it  mixes  in  all 
proportions,  also  with  alcohol  and  ether.  The  most  important  reactions 
for  acetone  are  the  following : 

Lieben's  Iodoform  Test.  When  a  watery  solution  of  acetone  is  treated 
with  alkali  and  then  with  some  iodo-potassium-iodide  solution  and  gently 
warmed  a  yellow  precipitate  of  iodoform  is  formed,  which  is  known  by  its 
odor  and  by  the  appearance  of  the  crystals  (six-sided  plates  or  stars)  under 
the  microscope.  This  reaction  is  very  delicate,  but  it  is  not  characteristic 
of  acetone.  Gunning's  modification  of  the  iodoform  test  consists  in  using 
an  alcoholic  solution  of  iodine  and  ammonia  instead  of  the  iodine  dissolved 
in  potassium  iodide  and  alkali  hydrate.  In  this  case,  besides  iodoform,  a 
black  precipitate  of  iodide  of  nitrogen  is  formed,  but  this  gradually  dis- 
appears on  standing,  leaving  the  iodoform  visible.  This  modification  has 
the  advantage  that  it  does  not  give  any  iodoform  with  alcohol  or  aldehyde. 
On  the  other  hand,  it  is  not  quite  so  delicate,  but  still  it  detects  0.01  milli- 
gram of  acetone  in  1  c.  c. 

Reynolds's  mercuric-oxide  test  is  based  on  the  power  of  acetone  to  dis- 
solve freshly  precipitated  HgO.  A  mercuric-chloride  solution  is  precipi- 
tated by  alcoholic  caustic  potash.  To  this  add  the  liquid  to  be  tested, 
shake  well,  and  filter.  In  the  presence  of  acetone  the  filtrate  contains 
mercury,  which  may  be  detected  by  ammonium  sulphide.  This  test 
has  about  the  same  delicacy  as  Gunning's  test.  Aldehydes  also  dissolve 
appreciable  quantities  of  mercuric  oxide. 

Legal's  Sodium  Nitroprusside  Test.  If  an  acetone  solution  is  treated 
with  a  few  drops  of  a  freshly  prepared  sodium  nitroprusside  solution  and 
then  with  caustic-potash  or  soda  solution,  the  liquid  is  colored  ruby-red. 
Creatinine  gives  the  same  color;  but  if  he  mixture  is  saturated  with  acetic 
acid,  the  color  becomes  carmine  or  purplish  red  in  the  presence  of  acetone, 
but  yellow  and  then  gradually  green  and  blue  in  the  presence  of  creatinine. 
With    this   test    paracresol    responds  with   a  reddish-yellow  color,    which 

1  Azemar,  "  Acctonurie  experimentale. ' '  Travaux  de  physiologie,  1898  (laboratoire 
de  M.  le  professeur  E.  H<klon,  Montpellier). 


576  URINE. 

becomes  light  pink  when  acidified  with  acetic  acid  and  cannot  be  mistaken 
for  acetone.  If  ammonia  is  employed  instead  of  the  caustic  alkali 
(Le  Nobel),  the  reaction  takes  place  with  acetone  but  not  with  aldehyde. 
Pexzoldt  's  indigo  test  depends  on  the  fact  that  orthonitrobenzaldehyde 
in  alkaline  solution  with  acetone  yields  indigo.  A  warm  saturated  and  then 
cooled  solution  of  the  aldehyde  is  treated  with  the  liquid  to  be  tested  for 
acetone  and  next  with  caustic  soda.  In  the  presence  of  acetone  the  liquid 
first  becomes  yellow,  then  green,  and  lastly  indigo  separates;  and  this  may 
be  dissolved  with  a  blue  color  by  shaking  with  chloroform.  1.6  milligrams 
acetone  can  be  detected  by  this  test. 

Bela  v.  Bitt6  's  l  reaction  is  based  on  the  fact  that  on  adding  a  solution  of 
metadinitrobenzene,  made  alkaline  with  caustic  potash,  to  acetone,  a  violet-red 
color  is  produced  which  becomes  cherry-red  on  acidifying  with  an  organic  acid  or 
metaphosphoric  acid.  Aldehyde  gives  a  similar  violet-red  color  which  becomes 
yellowish-red  on  acidification.     Creatinine  does  not  give  this  reaction. 

CH3 

CO 
Acetoacetic    acid,     C4H603,    acetylacetic    acid,   diacetic   acid  =  pTT 

COOH 
This  acid  has  not  been  observed  as  a  physiological  constituent  of  the  urine. 
It  occurs  in  the  urine  chiefly  under  the  same  conditions  as  acetone.  Like 
acetone  the  acetoacetic  acid  occurs  often  in  children,  especially  in  high 
fevers,  acute  exanthema,  etc.  Diacetic  acid  decomposes  readily  into 
acetone.  According  to  Araki  2  it  is  probably  produced  as  an  intermediate 
product  in  the  oxidation  of  /?-oxybutyric  acid  in  the  organism.  The  three 
bodies  appearing  in  the  urine,  acetone,  acetoacetic  acid,  and  /?-oxybutyric 
acid,  stand  in  close  relationship  to  each  other. 

This  acid  is  a  colorless,  strongly  acid  liquid  which  mixes  with  water, 
alcohol,  and  ether  in  all  proportions.  On  heating  to  boiling  with  water, 
and  especially  with  acids,  this  acid  decomposes  into  carbon  dioxide  and 
acetone,  and  therefore  gives  the  above-mentioned  reactions  for  acetone. 
It  differs  from  acetone  in  that  it  gives  a  violet-red  or  brownish-red  color 
with  a  dilute  ferric-chloride  solution.  For  the  detection  of  this  acid  we 
make  use  of  the  following  reactions  which  may  be  applied  directly  to  the 
urine. 

Gerhardt's  Reaction.  Treat  10-15  c.  c.  of  the  urine  with  ferric-chloride 
solution  until  it  fails  to  give  a  precipitate,  filter,  and  add  some  more  ferric 
chloride.  In  the  presence  of  acetoacetic  acid  a  wine-red  color  is  obtained. 
The  color  becomes  paler  at  the  room  temperature  within  twenty-four 
hours,  but  more  quickly  on  boiling  (differing  from  salicylic  acid,  phenol, 

1  Annal.  d.  Chem  u.  Pharm.,  269. 

2  Zeitschr.  f.  physiol.  Chem.,  18. 


DETECTION  OF  ACETONE   AND   ACETO ACETIC  ACID.  577 

Bulphocyanidee).     A  portion  of  the  urine  slightly  acidified  and  boiled  'Iocs 

do1  Lrivc  this  reaction  on  account  of  the  decomposition  of  the  acetoacetic 
acid. 

Arnold  cmd  Lipliawskt's  Junction.    6  c.  c.  of  a  solution  containing 

1  grain  of  />-aniiuoacetophcnonc  and  2  c.  c.  of  concentrated  hydrochloric  acid 
in  100  c.  c.  of  water  are  mixed  with  3  c.  c.  of  a  1  per  cent  potassium-nitrite 
solution  and  then  treated  with  an  equal  volume  of  urine.  A  few  drops 
of  concentrated  ammonia  are  now  added  and  violently  shaken.  A  brick- 
red  coloration  is  obtained.  Then  take  10  drops  to  2  c.  c.  of  this  mixture 
(according  to  the  quantity  of  acetoacetic  acid  in  the  urine),  add  15-20  c.  c. 
HC1  of  sp.  gr.  1.19,  3  c.  c.  of  chloroform,  and  2-4  drops  of  ferric-chloride  solu- 
tion and  mix  without  shaking.  In  the  presence  of  acetoacetic  acid  the 
chloroform  is  colored  violet  or  blue  (otherwise  only  yellowish  or  faintly 
red).  This  reaction  is  more  delicate  than  the  preceding  test  and  reacts 
with  0.04  p.  m.  acetoacetic  acid.  Large  amounts  of  acetone  (but  not  the 
quantity  occurring  in  urines)  give  this  reaction  according  to  Allard.1 

Detection  of  Acetone  and  Acetoacetic  Acid  in  the  Urine.  Before  testing 
for  acetone  test  for  acetoacetic  acid;  as  this  acid  gradually  decomposes  on 
allowing  the  urine  to  stand,  the  specimen  must  be  as  fresh  as  possible.  In 
the  presence  of  acetoacetic  acid  the  urine  gives  the  above-mentioned  tests. 
In  testing  for  acetone  in  the  presence  of  acetoacetic  acid  make  the  urine 
slightly  alkaline  and  shake  in  a  separatory  funnel  with  ether  free  from 
alcohol  and  acetone.  Remove  the  ether  and  shake  it  with  water,  which 
takes  up  the  acetone  and  test  for  acetone  in  the  watery  solution.' 

In  the  absence  of  acetoacetic  acid  the  acetone  may  be  tested  for 
directly  in  the  urine;  this  may  be  done  by  Penzoldt's  test.  This  test, 
which  is  only  approximate,  is  of  value  only  when  the  urine  contains  a 
considerable  amount  of  acetone.  For  a  more  accurate  test  we  distill  at 
least  250  c.  c.  of  the  urine  faintly  acidified  with  sulphuric  acid,  care  being 
taken  to  have  a  good  condensation.  Most  of  the  acetone  is  contained  in 
the  first  10-20  c.  c.  of  the  distillate.  A  sure  result  may  be  obtained  by 
distilling  a  large  quantity  of  urine  until  about  TV  has  been  distilled  off. 
acidify  the  distillate  with  hydrochloric  acid,  redistill  and  repeat  this 
several  times,  collecting  the  first  portion  of  each  distillation.  The  final 
distillate  is  used  for  the  above  reactions.2 

The  quantitative  estimation  of  acetone  in  the  urine  is  done  by  converting 
it  first  into  iodoform.  The  urine  is  acidified  with  acetic  acid  (according  to 
Huppert,  1-2  c.  c.  50  per  cent  acetic  acid  for  every  100  c.  c.  urine)  and 
distilled.  The  quantity  of  acetone  in  the  distillate  is  best  determined 
according  to  Messixger  and  Huppert's  method  by  determining  volu- 
metrically  the  quantity  of  iodine  used  in  the  formation  of  iodoform.  In 
regard  to  this  method  and  its  execution  the  reader  is  referred  to  Huppert- 
Nbubatjbr.' 

1  Arnold,   Wien.    klin.    Woehenschr.,    1S99,   and   Centralbl.    f.    innere  Med.,    1900; 
Lipliawsky,  Deutsch.  mod.  Woehenschr.,  1901;   Allard,  Berl.  klin.  Woehenschr.,  1901. 
7  See  also  Salkowski,  Pfliiger'a  Arch.,  56. 
* Harnanalyse,  760,  and  also  Geelmuyden,  Zeitschr.  f.  anal.  Chem.,  35. 


578  URINE. 

CH3 
/?-Oxybutyric  Acid,  C4H803=CHOH.    The  appearance  of  this  acid  in  the 

CH2 

COOH 
urine  was  first  positively  shown  by  Minkowski,  Kulz,  and  Stadelmann.1 
It  occurs  especially  in  severe  cases  of  diabetes,  when  it  may  form  the  largest 
portion  of  the  acetone  bodies  (Magnus-Levy,  Geelmuyden).  It  has  also 
been  observed  in  scarlet  fever,  measles,  in  scurvy,  and  in  diseases  of  the  brain 
with  abstinence.  It  seems  to  be  always  accompanied  with  acetoacetic  acid. 
The  /?-oxybutyric  acid  ordinarily  forms  an  odorless  syrup,  but  may  also 
be  obtained  as  crystals.  It  is  readily  soluble  in  water,  alcohol,  and  ether. 
It  is  lsevorotatory ;  («)d=  —24.12°  for  solutions  of  1-11  per  cent  and  has 
a  disturbing  action  upon  the  determination  of  sugar  by  means  of  the 
polariscope.  It  is  not  precipitated  by  basic  lead  acetate  or  by  ammoniacal 
lead  acetate,  neither  does  it  ferment.  On  boiling  with  water,  'especially  in 
the  presence  of  a  mineral  acid,  this  acid  decomposes  into  a-crotonic  acid,. 
which  melts  at  71-72°  C,  and  water:  CH3.CH(OH).CH2.COOH  =  H20  + 
CH3.CH:CH.COOH.  It  yields  acetone  on  oxidation  with  a  chromic-acid 
mixture. 

Detection  of  (3-Oxybutyric  Acid  in  the  Urine.  If  a  urine  is  still  Isevo- 
gyrate  after  fermentation  with  yeast,  the  presence  of  oxybutyric  acid  is 
probable.  A  further  test  may  be  made,  according  to  Kulz,  by  evaporating 
the  fermented  urine  to  a  syrup,  and,  after  the  addition  of  an  equal  volume 
of  concentrated  sulphuric  acid,  distilling  directly  without  cooling,  a-cro- 
tonic acid  is  produced  which  distills  over,  and,  after  collecting  in  a  test- 
tube,  crystals,  which  melt  at  +72°  C,  separate  on  cooling.  If  no  crystals 
are  obtained,  shake  the  distillate  with  ether,  evaporate,  and  test  the  melt- 
ing-point of  the  residue  which  has  been  washed  with  the  water.  Accord- 
ing to  Minkowski  the  acid  may  be  isolated  as  a  silver  salt.2 

The  Quantitative  Estimation  may  be  performed  as  follows,  according 
to  Bergell:  3  100-300  c.  c.  of  the  sugar-free  urine  or  fermented  urine 
is  made  slightly  alkaline  with  sodium  carbonate  and  concentrated  to  a 
syrup.  This,  on  cooling,  is  rubbed  with  syrupy  phosphoric  acid  (keeping 
it  cool),  anhydrous  copper  sulphate  (20-30  grams),  and  fine  sand,  and  the 
dry  mass  thoroughly  extracted  with  anhydrous  ether  in  an  extraction 
apparatus.  The  residue  after  the  evaporation  of  the  ether  is  dissolved 
in  water  and  decolorized,  if  necessary,  with  animal  charcoal,  and  the  quan- 
tity of  the  acid  calculated  from  the  polarization.  Other  methods  have  been 
suggested  by  Darmstadter,  Boekelman  and  Bouma.4 

'Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  18  and  19;  Stadelmann,  ibid.,  17; 
Kiilz,  Zeitschr.  f.  Biologie,  20  and  23. 

2  Arch.  f.  exp.  Path.  u.  Pharm.,  18,  35;  Zeitschr.  f.  anal.  Chem.,  24,  153. 

3  Zeitschr.  f.  physiol.  Chem.,  33. 

*  Darmstadter,  ibid.,  37;  Boekelman  and  Bouma,  see  Maly's  Jahresber.,  31. 


DIAZO  REACTION.  579 

Ehruch's  l  Urine  Test.  Mix  250  c.  c.  of  a  solution  which  contains  50  c.  c 
IK  '1  and  l  gram  of  Bulphanilic  acid  in  one  liter  with  5  <■.  c.  of  a  J  per  cent  solution 

of   Bodium    nitrite  (which   produces   very   little   of   the  active   body,  sulphodiazo- 

:iei.     In  performing  this  test  treat  the  urine  with  an  equal  volume  of  this 

mixture   and   then   supersaturate   with   ammonia,     Normal   urine   will   become 

yellow  thereby,  or  orange  after  the  addition  of  ammonia  (aromatic  oxyacids  may 

times  after  a  certain  time  give  reel  azo  bodies  which  color  the  upper  layer  of 
the  phosphate  sediment  |.  In  pathological  urines  there  sometimes  occurs  (and  this 
IS  the  characteristic  diazo  reaction)  a  primary  yellow  coloration,  with  a  very 
marked  secondary  red  coloration  on  the  addition  of  ammonia,  and  the  froth  is 
also  tinged  with  red.  The  upper  layer  of  the  sediment  becomes  greenish.  The 
body  which  gives  this  reaction  is  unknown,  but  it  occurs  especially  in  the  urine 
of  typhoid  patients  (Eiiruch).  Opinions  differ  in  regard  to  the  significance  of 
this  reaction.  The  fact  that  the  oxyproteic  acid  gives  this  reaction  as  above 
stated    page  524)  is  of  inte  est. 

Another  urine  test  suggested  by  Ehrlich  consists  in  adding  a  hydrochloric 
acid  containing  2  per  cent  dlmethylaminobenzaldehyde  to  the  urine;  normal  urines 
are  colored  faintly  red,  while  certain  pathological  urines  become  cherry-red. 
This  reaction  (whose  cause  is  not  known2)  has  still  no  practical  importance. 

Rosentba.Ch'8  urine  test,  which  consists  in  adding  nitric  acid  drop  by  drop 
to  the  boiling-hot  urine  and  obtaining  a  claret-red  coloration  and  a  bluish-red 
foam  on  shaking,  depends  upon  the  formation  of  indigo  substances,  especially 
indigo  red.3 

Fat  in  the  Urine.  The  elimination  of  a  urine  which  in  appearance  and  rich- 
ness in  fat  resembles  chyle  is  called  chyluria.  It  habitually  contains  protcid  and 
often  fibrin.  Chyluria  occurs  mostly  in  the  inhabitants  of  the  tropics.  lApuria, 
or  the  elimination  of  fat  with  the  urine,  may  appear  in  apparently  healthy  persons, 
sometimes  with  and  sometimes  without  albuminuria,  in  pregnancy,  and  also  in 
certain  diseases,  as  in  diabetes,  poisoning  with  phosphorus,  and  fatty  degeneration 
of  the  kidneys. 

Fat  is  usually  detected  by  the  microscope.  It  may  also  be  dissolved  with 
ether,  and  may  invariably  be  detected  by  evaporating  the  urine  to  dryness  and 
extracting  the  residue  with  ether. 

'<  stt  rin  is  also  sometimes  found  in  the  urine  in  chyluria  and  in  a  few  other 
eases. 

Leucin  and  Tyrosin.  These  bodies  are  found  in  the  urine,  especially 
in  acute  yellow  atrophy  of  the  liver,  in  acute  phosphorus-poisoning,  and 
in  severe  cases  of  typhoid  and  smallpox. 

Detection  of  Leucin  and  Tyrosin.  Tyrosin  occurring  as  a  sediment  may  be 
identified  by  means  of  the  microscope;  but  if  a  positive  proof  is  desired,  a  recrys- 
tallization  of  the  same  from  ammonia  or  ammoniacal  alcohol  is  necessary. 

To  detect  both  these  bodies  when  they  occur  in  solution  in  the  urine,  proceed 
in  the  following  manner:  The  urine  free  from  proteid  is  precipitated  by  basic  lead 
•  •.  the  lead  removed  from  the  filtrate  by  U.S.  and  the  solution  concentrated 
as  much  as  possible.  The  residue  is  extracted  with  a  small  quantity  of  absolute 
alcohol  to  remove  the  urea.  The  residue  is  then  boiled  with  faintly  ammoniacal 
alcohol,  filtered,  the  filtrate  evaporated  to  a  small  volume  and  allowed  to  crys- 
tallize.    If  no  tyrosin  crystals  are  obtained,  then  dilute  with  water,  precipitate 

1  Ehrlich,  Zeitsclir.  f.  klin.  Med.,  5.  See  also  Clemens,  Deutsch.  Arch.  f.  klin. 
Med..  l'»3  (literature). 

'Pee  Proscher,  Zeitschr.  f.  physioL  Chem.,  31,  and  Clemens,  Deutsch  Arch.  f. 
klin.  Med..  71. 

3  See  Rosin,  Virchow's  Arch.,  123. 


5S0  URINE. 

again  with  basic  lead  acetate,  and  proceed  as  before.  If  tyrosin  crystals  now 
separate,  they  are  filtered,  and  the  filtrate  still  further  concentrated  to  obtain 
the  leucin  crystals. 

Cystin  (see  page  74).  Baumann  and  Goldmann  *  claim  that  a  sub- 
stance similar  to  cystin  occurs  in  very  small  amounts  in  normal  urine. 
This  substance  occurs  in  large  quantities  in  the  urine  of  dogs  after  poison- 
ing with  phosphorus.  Cystin  itself  is  only  found  with  positivene'ss,  and 
even  then  very  rarely,  in  urinary  calculi  and  in  pathological  urines,  from 
which  it  may  separate  as  a  sediment.  Cystinuria  occurs  oftener  in  men 
than  in  women.  Baumann  and  v.  Tjdranszky  found  in  urine  in  cystinuria 
the  two  diamines,  cadaverin  (pentamethylendiamine)  and  putrescin  (tetra- 
methylendiamine),  which  are  produced  in  the  putrefaction  of  proteids. 
These  two  diamines  were  also  found  in  the  contents  of  the  intestine  in 
cystinuria,  while  under  normal  conditions  they  are  not  present.  Hammar- 
sten  therefore  considers  that  perhaps  some  connection  exists  between 
the  formation  of  diamines  in  the  intestine,  by  the  peculiar  putrefaction 
in  cystinuria,  and  cystinuria  itself.  Cases  of  cystinuria,  even  without 
diamines  in  the  urine,  have  recently  been  observed  and  reported  by  other 
investigators.  Diamines  are  only  seldom  found  in  the  urine  as  well  as 
in  the  faeces,  which  perhaps  depends  upon  the  fact,  observed  by  Cammidge 
and  Garrod,  that  the  diamines  occur  only  occasionally  in  the  fasces.  The 
properties  and  reactions  of  cj^stin  have  been  given  on  page  75. 

Cystin  is  easily  prepared  from  cystin  calculi  by  dissolving  them  in 
alkali  carbonate,  precipitating  the  solution  with  acetic  acid,  and  redissolv- 
ing  the  precipitate  in  ammonia.  The  cystin  crystallizes  on  the  spontane- 
ous evaporation  of  the  ammonia.  The  cystin  dissolved  in  the  urine  is 
detected,  in  the  absence  of  proteid  and  sulphuretted  hydrogen,  by  boiling  with 
alkali  and  testing  with  a  lead  salt  or  sodium  nitroprusside.  To  isolate  cystin 
from  the  urine,  acidify  the  urine  strongly  with  acetic  acid.  The  precipitate 
containing  cystin  is  collected  after  twenty-four  hours  and  digested  with 
hydrochloric  acid,  which  dissolves  the  cystin  and  calcium  oxalate,  leaving 
the  uric  acid  undissolved.  Filter,  supersaturate  the  filtrate  with  ammo- 
nium carbonate,  and  treat  the  precipitate  with  ammonia,  which  dissolves 
the  cystin  and  leaves  the  calcium  oxalate.  Filter  again  and  precipitate 
with  acetic  acid.  The  precipitated  cystin  is  identified  by  the  microscope 
and  the  above-mentioned  reactions.  Cystin  as  a  sediment  is  identified  by 
the  microscope.  It  must  be  purified  by  dissolving  in  ammonia  and  pre- 
cipitating with  acetic  acid  and  then  tested.  Traces  of  dissolved  cystin  may 
be  detected  by  the  production  of  benzoyl-cystin,  according  to  Baumann 
and  Goldmann. 

1  Baumann,  Zeitschr.  f.  physiol.  Chem.,  8.  In  regard  to  the  literature  on  cystin 
see  Brenzinger,  ibid.,  16;  Baumann  and  Goldmann,  ibid.,  12;  Baumann  and  v. 
Tdnlnszky,  ibid.,  13;  Stadthagen  and  Bneger,  Berlin,  klin.  Wochenschr. ,  1889;  Cam- 
midge and  Garrod,  Journ.  of  Path.  u.  Bacteriol.  1900  (literature  on  the  diamines  in 
the  urine  and  feces). 


URINARY  SEDIMENTS.  581 

VII.   Urinary  Sediments  and  Calculi. 

Urinary  sediment  is  the  more  or  less  abundant  deposit  which  is  found 
in  the  urine  after  standing.  This  deposit  may  consist  partly  of  organized 
and  partly  of  non-organized  constituents.  The  first,  consisting  of  celLs  of 
various  kinds,  yeast-fungi,  bacteria,  spermatozoa,  casts,  etc.,  must  be 
investigated  by  means  of  the  microscope,  and  the  following  only  applies 
to  the  non-organized  deposits. 

As  previously  mentioned  (page  462),  the  urine  of  healthy  individuals  may 
sometimes,  even  on  voiding,  be  cloudy  on  account  of  the  phosphates  present, 
or  become  so  after  a  little  while  because  of  the  separation  of  urates.  As  a 
rule  urine  just  voided  is  clear,  and  after  cooling  shows  only  a  faint  cloud 
(nubecula)  which  consists  of  urine  mucoid,  a  few  epithelium-cells,  mucous 
corpuscles,  and  urate  particles.  If  an  acid  urine  is  allowed  to  stand,  it  will 
gradually  change;  it  becomes  darker  and  deposits  a  sediment  consisting  of 
uric  acid  or  urates,  and  sometimes  also  calcium-oxalate  crystals,  in  which 
yeast -fungi  and  bacteria  are  often  to  be  seen.  This  change,  which  the 
earlier  investigators  called  "acid  fermentation  of  the  urine,"  is  gener- 
ally considered  as  an  exchange  of  the  dihydrogen  alkali  phosphates  with 
the  urates  of  the  urine.  Monohydrogen  phosphates  besides  acid  urates 
or  free  uric  acid  or  a  mixture  of  both,  according  to  conditions,1  are  hereby 
formed, 

Sooner  or  later,  sometimes  only  after  several  weeks,  the  reaction  of  the 
original  acid  urine  changes  and  becomes  neutral  or  alkaline.  The  urine  has 
now  passed  into  the  "alkaline  fermentation,"  which  consists  in  the 
decomposition  of  the  urea  into  carbon  dioxide  and  ammonia  by  means  of 
lower  organisms,  micrococcus  ureae,  bacterium  urese,  and  other  bacteria. 
Musculus  2  has  isolated  an  enzyme  from  the  micrococcus  urese  which 
decomposes  urea  and  is  soluble  in  water.  During  the  alkaline  fermentation 
volatile  fatty  acids,  especially  acetic  acid,  may  be  produced,  chiefly  by 
the  fermentation  of  the  carbohydrates  of  the  urine  (Salkowski  3).  A 
fermentation  by  which  nitric  acid  is  reduced  to  nitrous  acid,  and  another 
where  sulphuretted  hydrogen  is  produced,  may  sometimes  occur. 

When  the  alkaline  fermentation  has  advanced  only  so  far  as  to  render 
the  reaction  neutral,  there  often  occurs  in  the  sediment  fragments  of  uric- 
acid  crystals,  sometimes  covered  with  prismatic  crystals  of  alkali  urate; 
dark-colored  spheres  of  ammonium  urate,  crystals  of  calcium  oxalate,  and 
sometimes  crystallized  calcium  phosphate  are  also  found.  Crystals  of 
ammonium-magnesium  phosphate  (triple  phosphate)  and  spherical  ammo- 
nium  urate   are   specially    characteristic   of   alkaline   fermentation.     The 

'See  Huppert-Xeubauer,  10.  Aufl.,  and  A.  Ritter,  Zeitschr.  f.  Biologie,  35. 

2  Musculus,  Pfliiger's  Arch.,  12. 

3  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  13. 


5S2  URINE. 

urine  in  alkaline  fermentation  becomes  paler  and  is  often  covered  with  a 
fine  membrane  which  contains  amorphous  calcium  phosphate  and  glisten- 
ing crystals  of  triple  phosphate  and  numerous  micro-organisms. 

Non-organized  Sediments. 

Uric  Acid.  This  acid  occurs  in  acid  urines  as  colored  crystals  which  are 
identified  partly  by  their  form  and  partly  by  their  property  of  giving  the 
murexid  test.  On  warming  the  urine  they  are  not  dissolved.  On  the 
addition  of  caustic  alkali  to  the  sediment  the  crystals  dissolve,  and  when  a 
drop  of  this  solution  is  placed  on  a  microscope-slide  and  treated  with  a  drop 
of  hydrochloric  acid,  small  crystals  of  uric  acid  are  obtained  which  can  be 
easily  seen  under  the  microscope. 

Acid  Urates.  These  occur  only  in  the  sediment  of  acid  or  neutral 
urines.  They  are  amorphous,  clay -yellow,  brick-red,  rose-colored,  or 
brownish-red.  They  differ  from  other  sediments  in  that  they  dissolve  on 
warming  the  urine.  They  give  the  murexid  test,  and  small  microscopic 
crystals  of  uric  acid  separate  on  the  addition  of  hydrochloric  acid.  Crys- 
talline alkali  urates  occur  very  rarely  in  the  urine,  and  as  a  rule  only  in 
such  as  have  become  neutral  but  not  alkaline  by  alkaline  fermentation. 
The  crystals  are  somewhat  similar  to  those  of  neutral  calcium  phosphate; 
they  are  not  dissolved  by  acetic  acid,  however,  but  give  a  cloudiness  there- 
with due  to  small  crystals  of  uric  acid. 

Ammonium  urate  may  indeed  occur  as  a  sediment  in  a  neutral  urine 
which  at  first  was  strongly  acid  and  has  become  neutralized  by  the  alkaline 
fermentation,  but  it  is  only  characteristic  of  ammoniacal  urines.  This 
sediment  consists  of  yellow  or  brownish  rounded  spheres  which  are  often 
covered  with  thorny-shaped  prisms  and,  because  of  this,  are  rather  large 
and  resemble  the  thorn-apple.  It  gives  the  murexid  test.  It  is  dissolved 
by  alkalies  with  the  development  of  ammonia,  and  crystals  of  uric  acid 
separate  on  the  addition  of  hydrochloric  acid  to  this  solution. 

Calcium  oxalate  occurs  in  the  sediment  generally  as  small,  shining, 
strondy  refractive  quadratic  octahedra,  which  on  microscopical  examina- 
tion remind  one  of  a  letter-envelope.  The  crj^stals  can  only  be  mistaken 
for  small,  not  fully  developed  crystals  of  ammonium-magnesium  phos- 
phate. They  differ  from  these  by  their  insolubility  in  acetic  acid.  The 
oxalate  may  also  occur  as  flat,  oval,  or  nearly  circular  disks  with  central 
cavities  which  from  the  side  appear  like  an  hour-glass.  Calcium  oxalate 
may  occur  as  a  sediment  in  an  acid  as  well  as  in  a  neutral  or  alkaline  urine. 
The  Quantity  of  calcium  oxalate  separated  from  the  urine  as  sediment 
depends  not  only  upon  the  amount  of  this  salt  present,  but  also  upon  the 
acidity  of  the  urine.  The  solvent  for  the  oxalate  in  the  urine  seems  to  be 
the  di-acid  alkali  phosphate,  and  the  greater  the  quantity  of  this  salt  in  the 
urine  the  greater  the  quantity  of  oxalate  in  solution.     When,  as  previously 


NON-ORGANIZED  SEDIMENTS.  583 

mentioned  (page  5S1),  the  simple-acid  phosphate  is  formed  from  the  di-acid 
phosphate,  on  allowing  the  urine  to  stand,  a  corresponding  part  of  the  oxa- 
late may  be  separated  as  sediment. 

Calcium  carbonate  occurs  in  considerable  quantities  as  sediment  in  the 
urine  of  herbivora.  It  occurs  in  but  small  quantities  as  a  sediment  in 
human  urine,  and  in  fact  only  in  alkaline  urines.  It  either  has  almost  the 
same  appearance  as  amorphous  calcium  oxalate  or  it  occurs  as  somewhat 
larger  spheres  with  concentric  bands.  It  dissolves  in  acetic  acid  with  the 
generation  of  gas,  which  differentiates  it  from  calcium  oxalate.  It  is  not 
yellow  or  brown  like  ammonium  urate,  and  does  not  give  the  murexid 
test. 

Calcium  sulphate  occurs  very  rarely  as  a  sediment  in  strongly  acid  urine.  It 
appears  as  long,  thin,  colorless  needles,  or  generally  as  plates  grouped  together. 

Calcium  Phosphate.  The  calcium  triphosphate,  Ca3(P04)2,  which 
occurs  only  in  alkaline  urines,  is  always  amorphous  and  occurs  partly  as  a 
colorless,  very  fine  powder  and  partly  as  a  membrane  consisting  of  very 
fine  granules.  It  differs  from  the  amorphous  urates  in  that  it  is  colorless, 
dissolves  in  acetic  acid,  but  remains  undissolved  on  warming  the  urine. 
Calcium  diphosphate,  CaHP04-f  2H20,  occurs  in  neutral  or  only  in  very 
faintly  acid  urine.  It  is  found  sometimes  as  a  thin  film  covering  the  urine 
and  sometimes  as  a  sediment.  In  crystallizing,  the  crystals  may  be  single, 
or  they  may  cross  one  another,  or  they  may  be  arranged  in  groups  of  color- 
less, wedge-shaped  crystals  whose  wide  end  is  sharply  defined.  These  crys- 
tals differ  from  crystalline  alkali  urates  in  that  they  dissolve  without  a 
residue  in  dilute  acids  and  do  not  give  the  murexid  test. 

Ammonium-magnesium  phosphate,  triple  phosphate,  may  separate 
from  an  amphoteric  urine  in  the  presence  of  a  sufficient  quantity  of  am- 
monium salts,  but  it  is  generally  characteristic  of  a  urine  which  is  ammo- 
niacal  through  alkaline  fermentation.  The  crystals  are  so  large  that  they 
may  be  seen  with  the  unaided  eye  as  colorless  glistening  particles  in  the 
sediment,  on  the  walls  of  the  vessel,  and  in  the  film  on  the  surface  of  the 
urine.  This  salt  forms  large  prismatic  crystals  of  the  rhombic  system 
(coffin-shaped)  which  are  easily  soluble  in  acetic  acid.  Amorphous  magne- 
sium triphosphate,  Mg3(P04)2,  occurs  with  calcium  triphosphate  in  urines 
rendered  alkaline  by  a  fixed  alkali.  Crystalline  magnesium  phosphate, 
M<r3(P04),+  22H20,  has  been  observed  in  a  few  cases  in  human  urine  (also 
in  horse 's  urine)  as  strongly  refractive,  long  rhombic  plates. 

Kyestein  is  the  film  which  appears  after  a  little  while  on  the  surface  of  the  urine. 
This  coating,  which  was  formerly  considered  as  characteristic  of  urine  in  preg- 
nancy, contains  various  elements,  such  as  fungi,  vibriones,  epithelium-cells,  etc. 
It  often  contains  earthy  phosphates  and  triple-phosphate  crystals. 

As  more  rare  sediments  we  find  eystin,  tyrosin,  hippuric  acid,  xanthine,  hcema- 
toidin.  In  alkaline  urine  blue  crystals  of  indigo  may  also  occur,  due  to  a  decom- 
position of  indoxyl-glucuronic  acid. 


584  URINE. 


Urinary  Calculi. 

Besides  certain  pathological  constituents  of  the  urine,  all  those  urin- 
ary constituents  which  occur  as  sediments  take  part  in  the  formation  of 
urinary  calculi.  Ebstein  *  considers  the  essential  difference  between  an 
amorphous  or  crystalline  sediment  in  the  urine  on  one  side  and  urinary 
sand  or  large  calculi  on  the  other  to  be  the  occurrence  of  an  organic  frame 
in  the  latter.  As  the  sediments  which  appear  in  normal  acid  urine  and  in 
a  urine  alkaline  through  fermentation  are  different,  so  also  are  the  urinary 
calculi  which  appear  under  corresponding  conditions. 

If  the  formation  of  a  calculus  and  its  further  development  take  place  in 
an  undecomposed  urine,  it  is  called  a  primary  formation.  If,  on  the  con- 
trary, the  urine  has  undergone  alkaline  fermentation  and  the  ammonia 
formed  thereby  has  given  rise  to  a  calculus  formation  by  precipitating 
ammonium  urate,  triple  phosphate,  and  earthy  phosphates,  then  it  is  called 
a  secondary  formation.  Such  a  formation  takes  place,  for  instance,  when 
a  foreign  body  in  the  bladder  produces  catarrh  accompanied  by  alkaline 
fermentation. 

We  discriminate  between  the  nucleus  or  nuclei — if  such  can  be  seen — 
and  the  different  layers  of  the  calculus.  The  nucleus  may  be  essentially 
different  in  different  cases,  for  quite  frequently  it  consists  of  a  foreign  body 
introduced  into  the  bladder.  The  calculus  may  have  more  than  one  nu- 
cleus. In  a  tabulation  made  by  Ultzmann  of  545  cases  of  urinary  calculi, 
the  nucleus  in  80.9  per  cent  of  the  cases  consisted  of  uric  acid  (and  urates); 
in  5.6  per  cent,  of  calcium  oxalate;  in  8.6  per  cent,  of  earthy  phosphates; 
in  1.4  per  cent,  of  cystin;   and  in  3.3  per  cent,  of  some  foreign  body. 

During  the  growth  of  a  calculus  it  often  happens  that,  for  some  reason 
or  other,  the  original  calculus-forming  substance  is  covered  with  another 
layer  of  a  different  substance.  A  new  layer  of  the  original  substance  may 
deposit  on  the  outside  of  this,  and  this  process  may  be  repeated.  In  this 
way  a  calculus  consisting  originally  of  a  simple  stone  may  be  converted  into 
a  so-called  compound  stone  with  several  layers  of  different  substances. 
Such  calculi  are  always  formed  when  a  primary  is  changed  into  a  secondary 
formation.  By  the  continued  action  of  an  alkaline  urine  containing  pus, 
the  primary  constituents  of  an  originally  primary  calculus  may  be  partly 
dissolved  and  be  replaced  by  phosphates.  Metamorphosed  urinary  calculi 
are  formed  in  this  way. 

Uric-acid  calculi  are  very  abundant.  They  are  variable  in  size  and 
form.  The  size  of  the  bladder-stone  varies  from  that  of  a  pea  or  bean  to 
that  of  a  goose-egg.  Uric-acid  stones  are  always  colored;  generally  they 
are  grayish-yellow,  yellowish-brown,  or  pale  red-brown.     The  upper  surface 

1  Die  Natur  und  Behandlung  der  Harnsteine.     Wiesbaden,  1884. 


URINARY  CALCULI.  585 

is  sometimes  entirely  even  or  smooth,  sometimes  rough  or  uneven.  Next 
to  the  oxalate  calculus,  the  uric-acid  calculus  is  the  hardest.  The  fractured 
surface  shows  regular  concentric,  unequally  colored  layers  which  may  often 
be  removed  as  shells.  These  calculi  are  formed  primarily.  Layers  of  uric 
acid  sometimes  alternate  with  other  layers  of  primary  formation,  most 
frequently  with  layers  of  calcium  oxalate.  The  simple  uric-acid  calculus 
leaves  very  little  residue  when  burnt  on  a  platinum  foil.  It  gives  the 
murexid  test,  but  there  is  no  material  development  of  ammonia  when 
acted  on  by  caustic  soda. 

Ammonium-uratc  calculi  occur  as  primary  calculi  in  new-born  or  nurs- 
ing infants,  rarely  in  grown  persons.  They  often  occur  as  a  secondary 
formation.  The  primary  stones  are  small,  with  a  pale-yellow  or  dark- 
yellowish  surface.  When  moist  they  are  almost  like  dough;  in  the  dry 
state  they  are  earthy,  easily  crumbling  into  a  pale  powder.  They  give  the 
murexid  test  and  develop  much  ammonia  with  caustic  soda. 

Calcium-oxalate  calculi  are,  next  to  uric-acid  calculi,  the  most  abundant. 
They  are  either  smooth  and  small  (hemp-seed  calculi)  or  larger,  of  the 
size  of  a  hen's  egg,  with  rough,  uneven  surface,  or  their  surface  is  covered 
with  prongs  (mulberry  calculi).  These  calculi  produce  bleeding  easily, 
and  therefore  they  often  have  a  dark-brown  surface  due  to  decomposed 
blood-coloring  matters.  Among  the  calculi  occurring  in  man  these  are  the 
hardest.  They  dissolve  in  hydrochloric  acid  without  developing  gas,  but 
are  not  soluble  in  acetic  acid.  After  gently  heating  the  powder,  it  dissolves 
in  acetic  acid  with  frothing.  With  more  intense  heat  it  becomes  alkaline, 
due  to  the  production  of  quicklime. 

Phosphate  Calculi.  These,  which  consist  mainly  of  a  mixture  of  the 
normal  phosphate  of  the  alkaline  earths  with  triple  phosphate,  may  be  very 
large.  They  are  as  a  rule  of  secondary  formation  and  contain  besides 
these  phosphates  also  some  ammonium  urate  and  calcium  oxalate.  These 
calculi  ordinarily  consist  of  a  mixture  of  three  constituents — earthy 
phosphate,  triple  phosphate,  and  ammonium  urate — surrounding  a  foreign 
body  as  a  nucleus.  Their  color  is  variable — white,  dingy  white,  pale  yel- 
low, sometimes  violet  or  lilac-colored  (from  indigo  red).  The  surface  is 
always  rough.  Calculi  consisting  of  triple  phosphate  alone  are  seldom 
found.  They  are  ordinarily  small,  with  granular  or  radiated  crystalline 
fracture.  Stones  of  mono-acid  calcium  phosphate  are  also  seldom  obtained. 
They  are  white  and  have  beautiful  crystalline  texture.  The  phosphatic 
calculi  do  not  burn  up,  the  powder  dissolves  in  acid  without  effervescence, 
and  the  solution  gives  the  reactions  for  phosphoric  acid  and  the  alkaline 
earths.  The  triple-phosphate  calculi  generate  ammonia  on  the  addition  of 
an  alkali. 

Calcium-carbonate  calculi  occur  chiefly  in  herbivora.  They  are  seldom  found 
in  man.  They  have  mostly  chalky  properties,  and  are  ordinarily  white.  They 
are  completely  or  in  great  part  dissolved  by  acids  with  effervescence. 


586  URINE. 

Cystin  calculi  occur  but  seldom.  They  are  o'  primary  formation,  of  various 
sizes,  sometimes  as  large  as  a  hen's  egg.  They  have  a  smooth  or  rough  surface, 
are  white  or  pale  yellow,  and  have  a  crystalline  fracture.  They  are  not  very 
hard  and  are  consumed  almost  entirely  on  the  platinum  foil,  burning  with  a  bluish 
flam?.     They  give  the  above-mentioned  reacti  ns  for  cystin. 

Xanthine  calculi  are  very  rarely  found.  They  are  also  of  primary  formation. 
They  vary  from  the  size  of  a  pea  to  that  of  a  hen's  egg.  They  are  whitish,  yel- 
lowish-brown or  cinnamon-brown  in  color,  of  medium  hardness,  with  amorphous 
fracture,  and  on  rubbing  appear  like  wax.  They  burn  up  completely  when 
heated  on  a  platinum  foil.  They  give  the  xanthine  reaction  with  nitric  acid  and 
alkali  but  this  must  not  be  mistaken  for  the  murexid  test. 

Urostealith  calculi  have  been  observed  only  a  few  times.  In  the  moist  state 
they  are  soft  and  elastic  at  the  temperature  of  the  body,  but  in  the  dry  state  they 
are  brittle,  with  an  amorphous  fracture  and  waxy  appearance.  They  burn  with 
a  luminous  flame  when  heated  on  platinum  foil  and  generate  an  odor  similar  to 
resin  or  shellac.  Such  a  calculus,  investigated  by  Krukenberg,1  consisted  of 
paraffine  derived  from  a  parafhne  bougie  used  as  a  sound  on  the  patient.  Perhaps 
the  urostealith  calculi  observed  in  other  cases  had  a  similar  origin,  although  the 
substances  of  which  they  consisted  have  not  been  closely  studied.  Horbaczew- 
ski  has  recently  analyzed  a  case  of  urostealith  which,  to  all  appearances,  was 
formed  in  the  bladder.  This  calculus  contained  25  p.  m.  water,  8  p.  m.  inorganic 
bodies,  117  p.  m.  bodies  insoluble  in  ether,  and  850  p.  m„  organic  bodies  soluble 
in  ether,  among  which  were  515  p.  m.  free  fatty  acids,  335  p.  m.  fat,  and  traces  of 
cholesterin.  The  fatty  acids  consisted  of  a  mixture  of  stearic,  palmitic,  and 
probably  myristic  acids. 

Horbaczewski  2  has  also  analyzed  a  bladder  stone  which  contained  958.7  p.  m. 
cholesterin. 

Fibrin  calculi  sometimes  occur.  They  consist  of  more  or  less  changed  fibrin 
coagulum.     On  burning  they  develop  an  odor  of  burnt  horn. 

The  chemical  investigation  of  urinary  calculi  is  of  great  practical  impor- 
tance. To  make  such  an  examination  actually  instructive  it  is  necessary  to 
investigate  separately  the  different  layers  which  constitute  the  calculus. 
For  this  purpose  saw  the  calculus,  previously  wrapped  in  paper,  with  a  fine 
saw  so  that  the  nucleus  becomes  accessible.  Then  peel  off  the  different 
layers,  or,  if  the  stone  is  to  be  kept,  scrape  off  enough  of  the  powder  from 
each  layer  for  examination.  This  powder  is  then  tested  by  heating  on  the 
platinum  foil.  It  must  not  be  forgotten  that  a  calculus  is  never  entirely 
burnt  up,  and  also  that  it  is  never  so  free  from  organic  matter  that  on  heat- 
ing it  does  not  carbonize.  Do  not,  therefore,  lay  too  great  stress  on  a  very 
insignificant  unburnt  residue  or  on  a  very  small  amount  of  organic  matter, 
but  consider  the  calculus  in  the  former  case  as  completely  burnt  and  in  the 
latter  as  unaffected. 

When  the  powder  is  in  great  part  burnt  up,  but  a  significant  quantity  of 
unburnt  residue  remains,  then  the  powder  in  question  contains  as  a  rule 
urates  mixed  with  inorganic  bodies.  In  such  cases  remove  the  urate  with 
boiling  water  and  then  test  the  filtrate  for  uric  acid  and  the  suspected  bases. 
The  residue  is  then  tested  according  to  the  following  schema  of  Heller, 
which  is  well  adapted  to  the  investigation  of  urinary  calculi.  In  regard  to 
the  more  detailed  examination  the  reader  is  referred  to  special  works  on  the 
subject. 


1  Chem.  Untersuch.  z.  wissensch.  Med.,  2.     Cited  from  Maly's  Jahresber.,  19,  422. 

2  Zeitschr.  f.  physiol.  Chem.,  18. 


EXAMINATION  OF  CALCULI. 
On  heating  the  powder  on  platinum  foil,  it 


587 


Does  not  burn 


The  powder  when  treated  with 
HC1 


Does  not  effervesce 


The  powder  gently 
heated  with  HC1 


The  powder  when 

moistened  with  a 

little  KHO 


> 

tic 
EB 

2.2- 

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fa 
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33 

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re   "*» 


^  1 

p  § 

2  I 

3 

o  Bt 

3  33° 

P  o 


Does  burn 


With  flame 


3 

Kg 

2.2 

c  o 

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re    re 

B'o 

82 

3/3 

etc 
—  o 


3    ° 
re   ^ 

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re 


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The  powder 

gives  the 

murexid 

test 


The  powder 

when  treated 

with  KHO 

gives 


H 

o  *  =• 
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- 


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T3   3-a 

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o 


CHAPTER  XVI. 
THE  SKIN  AND  ITS  SECRETIONS. 

In  the  structure  of  the  skin  of  man  and  vertebrates  many  different 
kinds  of  substances  occur  which  have  already  been  considered,  such  as  the 
constituents  of  the  epidermal  formation,  the  connective  and  fatty  tissues, 
the  nerves,  muscles,  etc.  Among  these  the  different  horn  structures,  the 
hair,  nails,  etc.,  whose  chief  constituent,  keratin,  has  been  spoken  of  in 
another  chapter  (Chapter  II),  are  of  special  interest. 

The  cells  of  the  horny  structure  show,  in  proportion  to  their  age,  a 
different  resistance  to  chemical  reagents,  especially  fixed  alkalies.  The 
younger  the  horn-cell  the  less  resistance  it  has  to  the  action  of  alkalies; 
with  advancing  age  the  resistance  becomes  greater,  and  the  cell-membranes 
of  many  horn-formations  are  nearly  insoluble  in  caustic  alkalies.  Keratin 
occurs  in  the  horn  structure  mixed  with  other  bodies,  from  which  it  is 
isolated  with  difficulty.  Among  these  bodies  the  mineral  constituents  in 
many  cases  occupy  a  prominent  place  because  of  their  quantity.  Hair 
leaves  on  burning  5-70  p.  m.  ash,  which  may  contain  in  1000  parts  230 
parts  alkali  sulphates,  140  parts  calcium  sulphate,  100  parts  iron  oxide, 
and  even  400  parts  silicic  acid.  Dark  hair  on  burning  seems  generally, 
although  not  always,  to  yield  more  iron  oxide  than  blond.  The  nails  are 
rich  in  calcium  phosphate,  and  the  feathers  rich  in  silicic  acid,  which 
Drechsel  1  claims  exists  in  part  in  organic  combination  as  an  ester. 

According  to  Gautier  and  Bertrand  2  arsenic  also  occurs  in  the  epider- 
mal formations.  The  arsenic  is,  according  to  Gautier,  of  importance  in 
the  formation  and  growth  of  the  same,  and  on  the  other  hand  these  struc- 
tures, hair,  nails,  and  epidermis-cells,  are  of  great  importance  for  the 
excretion  of  arsenic. 

The  skin  of  invertebrates  has  been  the  subject,  in  a  few  cases,  of  chemi- 
cal investigation,  and  in  these  animals  various  substances  have  been 
found,  of  which  a  few,  though  little  studied,  are  worth  discussing.  Among 
these  bodies  tunicin,  which  is  found  especially  in  the  mantle  of  the  tunicata, 
and  the  widely  diffused  chitin,  found  in  the  cuticle-formation  of  inverte- 
brates, are  of  interest. 

'Centralbl.   f.   Physiol.,  11,  361. 

2  Gautier,  Compt.  rend.,  129,  130,  131;   Bertrand,  ibid.,  134. 

588 


TUNICIN.    CHITIN.  589 

Tunicin.  Cellulose  seems,  according  to  the  investigations  of  Ambronn,  to 
occur  rather  extensively  in  the  animal  kingdom  in  the  arthropoda  and  the  mol- 
lusks  It  has  been  known  for  a  long  time  as  the  mantle  of  the  tunicata,  and  this 
animal  cellulose  was  called  tunicin  by  Berthelot.  According  to  the  investiga- 
tions of  Wintkrstein  there  does  not  seem  to  exist  any  marked  difference  between 
tunicin  and  ordinary  vegetable  cellulose.  On  boiling  with  dilute;  acid  tunicin 
yields  dextrose,  as  shown  first  by  Francuimont *  and  later  confirmed  by  Win- 

TKKSTEIN. 

Chitin  is  not  found  in  vertebrates.  In  invertebrates  chitin  is  alleged 
to  occur  in  several  classes  of  animals;  but  it  can  be  positively  asserted 
that  true,  typical  chitin  is  found  only  in  articulated  animals,  in  which  it 
forms  the  chief  organic  constituent  of  the  shell,  etc.  According  to  Kraw- 
kow  2  chitin  of  the  shell,  etc.,  does  not  seem  to  occur  free,  but  in  com- 
bination with  another  substance,  probably  a  proteid-like  body.  Chitin 
also  occurs,  according  to  Gilson  and  Winterstein,3  in  certain  fungi. 

According  to  Sundvik  the  formula  of  chitin  is  probably  C60H100N8O38+ 
?i(H20),  where  n  may  vary  between  1  and  4,  and  it  is  probably  an  amine 
derivative  of  a  carbohydrate,  with  the  general  formula  ft(C12H20O10). 
According  to  Krawkow  the  chitins  of  different  origin  show  different  behavior 
with  iodine,  and  he  therefore  concludes  that  there  must  exist  quite  a  group 
of  chitins,  which  seem  to  be  amine  derivatives  of  different  carbohydrates, 
such  as  dextrose,  glycogen,  dextrins,  etc.  According  to  Zander  4  only  two 
chitins  exist,  one  of  which  turns  violet  with  iodine  and  zinc  chloride,  and 
the  other  brown. 

Chitin  is  decomposed  on  boiling  with  mineral  acids  and  yields,  as  showm 
by  Ledderhose,  glucosamine  and  acetic  acid.  Schmiedeberg  therefore 
considers  chitin  as  a  probable  acetyl  acetic-acid  combination  of  glucosamine. 
Frankel  and  Kelly,5  on  the  contrary,  consider  chitin  as  of  a  more  com- 
plicated composition.  The  most  characteristic  cleavage  product  obtained  by 
them  was  a  chitosamine  acetylized  at  the  nitrogen  atom,  C6H1205N.COCH3, 
and  a  second  product,  acetyldichitosamine,  C14H26O10N2,  wdiich  has  the 
same  composition  as  chitosan  (see  below),  but  is  essentially  different  in 
many  regards. 

In  the  dry  state  chitin  forms  a  white,  brittle  mass  retaining  the  form  of 
the  original  tissue.  It  is  insoluble  in  boiling  water,  alcohol,  ether,  acetic 
acid,  dilute  mineral  acids,  and  dilute  alkalies.     It  is  soluble  in  concentrated 

1  Ambronn,  Maly's  Jahresber.,  20;  Berthelot,  Annal.  de  Chim.  et  Phys.,  56,  Compt. 
rend.,  17;  Winterstein,  Zeitschr.  f.  physiol  Chem.,  18;  Franchimont,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  12. 

2  Zeitschr.  f.  Biologie,  29. 

'Gilson,  Compt.  rend.,  120;  Winterstein,  Ber.  d.  deutsch.  chem.  Gesellsch.,  27 
and  28. 

4  Sundvik,  Zeitschr.  f.  physiol.  Chem.,  5;   Zander,  Pfliiger's  Arch.,  66. 

s  Ledderhose,  Zeitschr.  f.  physiol.  Chem.,  2  and  4;  Schmiedeberg,  Arch.  f.  exp. 
Path.  u.  Pharm.,  28;   Frankel  and  Kelly,  Monatshefte  f.  Chem.,  23. 


590  THE  SKIN  AND  ITS  SECRETIONS. 

acids.  It  is  dissolved  without  decomposing  in  cold  concentrated  hydro- 
chloric acid,  but  is  decomposed  by  boiling  hydrochloric  acid.  When  chitin 
is  dissolved  in  concentrated  sulphuric  acid  and  the  solution  dropped  into 
boiling  water  and  then  boiled,  a  substance  is  obtained  (glucosamine,  chitos- 
amine)  which  reduces  copper  suboxide  in  alkaline  solutions.  According 
to  Hoppe-Seyler  and  Araki,1  on  heating  chitin  with  alkali  and  a  little 
water  to  180°  C.  a  cleavage  takes  place  with  the  splitting  off  of  acetic  acid, 
and  the  formation  of  a  new  substance,  chitosan,  C14H26N2O10,  which  retains 
the  shape  of  the  original  chitin.  Chitosan  is  insoluble  in  water  and  alkali, 
but  dissolves  in  dilute  acids,  also  acetic  acid,  and  is  colored  violet  by  a 
dilute  iodine  solution.  It  splits  into  acetic  acid  and  glucosamine  by  the 
action  of  hydrochloric  acid.  On  heating  with  acetic  anhydride  it  is  con- 
verted into  a  chitin-like  substance  which  is  not  identical  with  chitin  and 
contains  at  least  three  acetyl  groups.  According  to  Krawkow  the  various 
chitins  behave  differently  with  iodine  or  with  sulphuric  acid  and  iodine, 
in  that  some  are  colored  reddish  brown,  blue,  or  violet,  while  others  are 
not  colored  at  all. 

Chitin  may  be  easily  prepared  from  the  wings  of  insects  or  from  the 
shells  of  the  lobster  or  the  crab,  the  last-mentioned  having  first  been 
extracted  by  an  acid  so  as  to  remove  the  lime  salts.  The  wings  or  shells 
are  boiled  with  caustic  alkali  until  they  are  white,  afterward  washed  with 
water,  then  with  dilute  acid  and  water,  and  lastly  extracted  with  alcohol 
and  ether.  If  chitin  so  prepared  is  dissolved  in  cold,  concentrated  sulphuric 
acid  and  diluted  with  cold  water,  then  pure  chitin  separates  out,  having 
been  set  free  from  the  combination  with  the  other  bodies  (Krawkow). 

Hyalin  is  the  chief  organic  constituent  of  the  walls  of  hydatid  cysts.  From  a 
chemical  point  of  view  it  stands  close  to  chitin,  or  between  it  and  proteid.  In 
old  and  more  transparent  sacs  it  is  tolerably  free  from  mineral  bodies,  but  in 
younger  sacs  it  contains  a  great  quantity  (16  per  cent)  of  lime  salts  (carbonate, 
phosphate,  and  sulphate). 

According  to  Lucke  2  its  composition  is : 

C 

From  old  cysts 45 . 3 

From  young  cysts 44 . 1 

It  differs  from  keratin  on  the  one  hand  and  from  proteids  on  the  other  by  the 
absence  of  sulphur,  also  by  its  yielding,  when  boiled  with  dilute  sulphuric  acid,  a 
variety  of  sugar  in  large  quantities  (50  per  cent),  which  is  reducing,  fermentable, 
and  dextrogyrate.  It  differs  from  chitin  by  the  property  of  being  gradually 
dissolved  by  caustic  potash  or  soda,  or  by  dilute  acids;  also  by  its  solubility  on 
heating  with  water  to  150°  C. 

The  coloring  matters  of  the  skin  and  horn- formations  are  of  different 
kinds,  but  have  not  been  much  studied.  Those  occurring  in  the  stratum 
Malpighii  of  the  skin,  especially  of  the  negro,  and  the  black  or  brown  pig- 


^eitschr.  f.  physiol.  Chem.,  20.  2  Virchow's  Arch.,  19. 


H 

N 

O 

6.5 

5.2 

43.0 

6.7 

4.5 

44.7 

MEL  AN  INS.  591 

merit  occurring  in  the  hair,  belong  to  the  group  of  those  substances 
which  have  received  the  name  melanins. 

Melanins.  This  group  includes  several  different  varieties  of  amorphous 
black  or  brown  pigments  which  are  insoluble  in  water,  alcohol,  ether, 
chloroform,  and  dilute  acids,  and  which  occur  in  the  skin,  hair,  epithelium- 
cells  of  the  retina,  in  sepia,  in  certain  pathological  formations,  and  in  the 
blood  and  urine  in  disease.  Of  these  pigments  there  are  a  few,  such  as  the 
melanin  of  the  eye,  Schmiedeberg 's  sarcomelanin,  and  that  from  the 
melanotic  sarcomata  of  horses,  the  hippomclanin  (Nexcki,  Sieber,  and 
Berdez),  which  are  soluble  with  difficulty  in  alkalies,  while  others,  such  as 
the  pigment  of  the  hair  and  the  coloring  matter  of  certain  pathological 
swellings  in  man,  the  phyniatorhusin  (Nencki  and  Berdez),  are  easily  solu- 
ble in  alkalies.  The  humus-like  products,  called  melanoidic  acids  by 
Schmiedeberg,  obtained  on  boiling  proteids  with  mineral  acids,  are  rather 
easily  soluble  in  alkalies. 

Among  the  melanins  there  are  a  few,  for  example  the  choroid  pigment, 
which  are  free  from  sulphur  (Landolt  and  others) ;  others,  on  the  contrary, 
as  sarcomelanin  and  the  pigment  of  the  hair  and  of  horse-hair,  are  rather 
rich  in  sulphur  (2-4  per  cent),  while  the  phyniatorhusin  found  in  certain 
swellings  and  in  the  urine  (Nexcki  and  Berdez,  K.  Morxer)  is  very  rich 
in  sulphur  (8-10  per  cent).  Whether  any  of  these  pigments,  especially 
the  phyniatorhusin,  contains  any  iron  or  not  is  an  important  though  dis- 
puted point,  for  it  leads  to  the  question  whether  these  pigments  are  formed 
from  the  blood-coloring  matters.  K.  Morxer  and  later  also  Braxdl  and 
L.  Pfeiffer  found,  on  the  contrary,  that  this  pigment  did  contain  iron,  and 
they  consider  it  as  a  derivative  of  the  blood-pigments.  The  sarcomelanin 
(from  a  sarcomatous  liver)  analyzed  by  Schmiedeberg  contained  2.7  per 
cent  iron,  which  was  in  organic  combination  in  part  and  could  not  be  com- 
pletely removed  by  dilute  hydrochloric  acid.  The  sarcomclanic  acid  pre- 
pared by  Schmiedeberg  by  the  action  of  alkali  on  this  melanin  contained 
1.07  per  cent  iron.  The  sarcomelanin  investigated  by  Zdarek  and  v.  Zey- 
xek  '  also  contained  0.4  per  cent  iron. 

The  difficulties  which  attend  the  isolation  and  purification  of  the  mela- 
nins have  not  been  overcome  in  certain  cases,  while  in  others  it  is  question- 
able whether  the  final  product  obtained  has  not  another  composition  than 
the  original  coloring  matter,  owing  to  the  energetic  chemical  processes 
resorted  to  in  its  purification.  Under  such  circumstances  it  seems  that  a 
tabulation  of  the  analyses  of  different  melanin  preparations  made  up  to 
the  present  time  is  of  secondary  importance. 

1  Zeitschr.   f    physiol.  Chem.,  36.     The  literature  on  the  melanins  may  be  found 

in    Schmiedeberg,  " Elementarformeln    einiger   Eiweisskorper,    etc."  Arch.    f.    exp. 

Path.  u.  Pharm.,  39;  also  in  Kobert,  Wiener  Klinik,  28  (1901),  and  Spiegler,  Hof- 
meister's  Beitrage,  4. 


592  THE  SKIN  AND  ITS  SECRETIONS. 

The  one  or  more  pigments  of  the  human  hair  have  a  low  percentage 
of  nitrogen,  8.5  per  cent  (Sieber),  and  a  variable  but  considerable  amount 
of  sulphur,  2.71-4.10  per  cent.  The  great  quantity  of  iron  oxide  which 
remains  on  incinerating  hair  does  not  seem  to  belong  to  the  pigments. 
The  pigment  of  the  negro 's  skin  and  hair  was  found  entirely  free  from  iron 
by  Abel  and  Davis.  The  pigment  prepared  by  Spiegler  from  the  hair 
of  animals  also  contained  no  iron. 

So  little  is  known  about  the  structural  products  of  the  melanins  or 
melanoids  that  it  is  impossible  to  give  the  origin  of  these  bodies.  As 
undoubtedly  there  are  several  distinct  melanins,  their  origin  must  also  be 
distinct.  The  ferruginous  melanins  should  be  considered  as  originating 
from  the  blood-pigments  until  further  research  proves  otherwise.  Most 
melanins — and  this  is  also  true  for  the  melanoids  produced  from  proteids  on 
cleavage  with  acids  (Samuely) — yield  indol  or  skatol  and  a  pyrrol  substance, 
and  we  must  therefore  admit  with  Samuely  1  that  the  different  chromogen 
groups  contained  in  the  proteid  molecule,  which  readily  yield  aromatic 
and  specially  heterocyclic  nuclei,  condense  with  the  elimination  of  water 
and  absorption  of  oxygen,  producing  dark  products  the  mixture  of  which 
forms  the  melanoids. 

It  has  also  been  found  that  by  the  action  of  tyrosinases  upon  tyrosin 
dark  products  similar  to  melanin  are  formed,  and  these,  like  the  animal 
melanins,  yield  substances  smelling  like  skatol  on  fusion  with  alkali.  Cer- 
tain investigators,  such  as  Gessard,  v.  Furth  and  Schneider,2  are  there- 
fore of  the  opinion  that  tyrosin  is  the  mother-substance  of  the  melanins. 

In  addition  to  the  coloring  matters  of  the  human  skin  it  is  in  place  here  to 
treat  of  the  pigments  found  in  the  skin  or  epidermal  formation  of  animals. 

The  beautiful  color  of  the  feathers  of  many  birds  depends  in  certain  cases  on 
purely  physical  causes  (interference-phenomena),  but  in  other  cases  on  coloring 
matters  of  various  kinds.  Such  a  coloring  matter  is  the  amorphous  reddish-violet 
turacin,  which  contains  7  per  cent  copper  and  whose  spectrum  is  very  similar  to  that 
of  oxyhemoglobin.  Krukenberg  3  found  a  large  number  of  coloring  matters  in 
birds'  feathers,  namely,  zooerythrin,  zoojulvin,  turacovirdin,  zoorubin,  psittacofulvin, 
and  others  which  cannot  be  enumerated  here. 

Tetronerythrin,  so  named  by  Wurm,  is  a  red  amorphous  pigment  which  is 
soluble  in  alcohol  and  ether,  and  which  occurs  in  the  red  warty  spots  over  the  eyes 
of  the  heathcock  and  the  grouse,  and  which  is  very  widely  spread  among  the 
;n vertebrates  (Halliburton,  De  Merejkowski,  MacMunn).  Besides  tetron- 
erythrin MacMunn  found  in  the  shells  of  crabs  and  lobsters  a  blue  coloring  matter, 
cyanocrystallin,  which  turns  red  with  acids  and  by  boiling  water.  Hcematopor- 
phyrin,  according  to  MacMunn,4  also  occurs  in  the  integuments  of  certain  of  the 
lower  animals. 

1  Hofmeister's  Beitrage,  2. 

2  Gessard,  Compt.  rend.,  13fi;  v.  Furth  and  Schneider,  Hofmeister's  Beitrage,  1. 

3  Vergleichende  physiol.  Studien,  Abth.  5,  and  (2.  Reihe)  Abth.  1,  151,  Abth.  2,  1, 
and  Abth.  3,  128. 

4  Wurm,  cited  from  Maly's  Jahresber.,  1;  Halliburton,  Journ.  of  Physiol.,  6; 
Merejkowski,  Compt.  rend.,  93;  MacMunn,  Proc.  Roy.  Soc,  1883,  and  Journ.  of 
Physiol.,  7. 


si:  hum.  593 

In  certain  butterflies  (the  pieridine)  the  white  pigment  of  the  wings  consists, 
a>  :  Ihiwm  by  HOPKINS,1  of  uric  acid,  and  the  yellow  pigment  of  a  uric-acid  deriva- 
tive, lepidotic  acid,  which  yields  a  purple  substance,  lepidoporphyrin,  on  warming 
with  dilute  sulphuric  acid.  The  yellow  and  red  pigment  of  the  Vanessa  are, 
according  to  Linden,1  of  an  entirely  different  kind.  In  this  case  we  are  dealing 
with  a  compound  between  proteid  and  a  pigment  which  is  allied  to  bilirubin  or 
urobilin,  i.e.,  a  compound  similar  to  haemoglobin. 

In  addition  to  the  coloring  matters  thus  far  mentioned  a  few  others  found  in 
certain  animals  (though  not  in  the  skin)  will  be  spoken  of. 

Carminic  acid,  or  the  red  pigment  of  the  cochineal,  gives  on  oxidation,  according 
to    LlEBERMANN    and    VOSWINCKEL,"   cochtnillic  acid,   C,0II8O7,    and   roccinic  acid, 

(\,llso,,  the  first  being  the  tri-carbonic  acid,  and  the  other  the  di-carbonic  acid  of 
m-cresol.  The  beautiful  purple  solution  of  ammonium  carminate  has  two  absorp- 
tion-bands between  D  and  E  which  are  similar  to  those  of  oxyhemoglobin.  These 
bands  lie  nearer  to  E  and  closer  together  and  are  less  sharply  defined  Purple  is 
the  evaporated  residue  from  the  purple-violet  secretion,  caused  by  the  action 
of  the  sunlight,  from  the  so-called  "purple  gland"  of  the  mantle  of  certain  species 
of  murex  and  purpura.     Its  chemical  nature  has  not  been  investigated. 

Among  the  remaining  coloring  matters  found  in  invertebrates  may  be  men- 
tioned blue  stentorin,  actiniochrom,  bonellin,  poly  per  ytHrin,  pentacrinin,  antedonin, 
erustaceorvbin,  janthinin,  and  chlorophyll. 

Sebum  when  freshly  secreted  is  an  oily  semi-fluid  mass  which 
solidifies  on  the  upper  surface  of  the  skin,  forming  a  greasy  coating.  The 
quantity  varies  with  the  individual.  Hoppe-Seyler  has  found  in  the 
sebum  a  body  similar  to  casein  besides  albumin  and  fat.  Cholesterin  is 
also  found  in  this  fat,  and  in  especially  large  quantities  in  the  vernix 
caseosa.  The  solids  of  the  sebum  consist  chiefly  of  fat,  epithelium-cells, 
and  protein  bodies;  the  vernix  caseosa  is  made  up  chiefly  of  fat. 
RuppEL  4  found  on  an  average  in  the  vernix  caseosa  348.52  p.  m.  water 
and  138.72  p.  m.  ether  extractives.  Besides  cholesterin  he  found  also 
isocholesterin. 

On  account  of  the  generally  diffused  view  that  the  wax  of  the  plant 
epidermis  serves  as  protection  for  the  inner  parts  of  the  fruit  and  plant,  Lie- 
breich  5  has  suggested  that  these  combinations  of  fatty  acids  with  mona- 
tomic  alcohols  are  the  cause  of  the  waxes  having  a  greater  resistance  as  com- 
pared with  the  glycerine  fats.  He  also  considers  that  the  cholesterin  fats  play 
the  role  of  a  protective  fat  in  the  animal  kingdom,  and  he  has  been  able 
to  detect  cholesterin  fat  in  human  skin  and  hair,  in  vernix  caseosa,  whale- 
bone, tortoise-shell,  cow's  horn,  the  feathers  and  beaks  of  several  birds, 
the  spines  of  the  hedgehog  and  porcupine,  the  hoofs  of  horses,  etc.  He 
draws  the  following  conclusion  from  this,  namely,  that  the  cholesterin  fats 
always  appear  in  combination  with  the  keratinous  substance,  and  that 


'Phil.  Trans.,  186. 

3Pfluger's  Arch.,  98. 

'  Ber.  d.  deutsch.  chem.  Gesellsch.,  30. 

*  Hoppe-Seyler,  Physiol.  Chem.,  7G0;   Riippel,  Zeitschr.  f.  physiol.  Chem.,  2L 

1  Virchow  's  Arch. ,  121. 


594  THE  SKIN  AND  ITS  SECRETIONS. 

the  cholesterin  fat,  like  the  wax  of  plants,  serves  as  protection  for  the  skin- 
surface  of  animals. 

In  the  fatty  protective  substance  secreted  by  the  Psylla  alni  Sundvik1  has 
found  psylla-alcohol,  CggHegO,  which  exists  there  as  an  ester  in  combination  with 
psyllic  acid,  C^H^COOH. 

Cerumen  is  a  mixture  of  the  secretion  of  the  sebaceous  and  sweat  glands 
of  the  cartilaginous  part  of  the  outer  organs  of  hearing.  It  contains  chiefly 
soaps  and  fat,  fatty  acids,  cholesterin  and  proteid,  and  besides  these  a  red 
substance  easily  soluble  in  alcohol  and  with  a  bitter-sweet  taste.2 

The  preputial  secretion,  smegma  prceputii,  contains  chiefly  fat,  also 
cholesterin  and  ammonium  soaps,  which  probably  are  produced  from 
decomposed  urine.  The  hippuric  acid,  benzoic  acid,  and  calcium  oxalate 
found  in  the  smegma  of  the  horse  have  probably  the  same  origin. 

We  may  also  consider  as  a  preputial  secretion  the  castoreum,  which  is  secreted 
by  two  peculiar  glandular  sacs  in  the  prepuce  of  the  beaver.  This  castoreum  is  a 
mixture  of  proteids,  fat,  resins,  traces  of  phenol  (volatile  oil),  and  a  non-nitrog- 
enous body,  castorin,  crystallizing  in  four-sided  needles  from  alcohol,  insoluble 
in  cold  water,  but  somewhat  soluble  in  boiling  water,  and  whose  composition  is 
little  known. 

In  the  secretion  from  the  anal  glands  of  the  skunk  butyl  mercaptan  and  alkyl 
sulphide  have  been  found  (Aldrich,  E.  Beckmann  3). 

Wool-fat,  or  the  so-called  fat-sweat  of  sheep,  is  a  mixture  of  the  secretion  of 
the  sudoriparous  and  sebaceous  glands.  There  is  found  in  the  watery  extract  a  large 
quantity  of  potassium  which  is  combined  with  organic  acid,  volatile  and  non-volatile 
fatty  acids,  benzoic  acid,  phenol-sulphuric  acid,  lactic  acid,  malic  acid,  succinic 
acid,  and  others.  The  fat  ontains,  among  other  bodies,  abundant  quantities 
of  ethers  of  fatty  acids  with  cholesterin  and  isocholesterin.  Darmstadter  and 
Lifschutz  4  have  found  other  alcohols  in  wool-fat  besides  myristic  acid,  also 
two  oxyfatty  acids,  lanoceric  acid,  C30H60O4,  and  lanopalmitic  acid,  CigH^Og. 

The  secretion  of  the  coccygeal  glands  of  ducks  and  geese  contains  a  body  similar 
to  casein,  besides  albumin,  nuclein,  lecithin,  and  fat,  but  no  sugar  (De  Jonge). 
Poison  us  bodies  have  been  found  in  the  secretion  of  the  skin  of  the  salamander 
and  the  toad,  nimely,  samandarin  (Zaleski,  Faust)  and  bufidin  (Jornara  and 
Casali),  bufotalin  and  the  disputed  bodies  bufonin  and  bufotenin  (Faust,  Ber- 
trand  and  Phisalix5). 

The  Perspiration.  Of  the  secretions  of  the  skin,  whose  quantity  amounts 
to  about  -fa  of  the  weight  of  the  body,  a  disproportionally  large  part  consists 
of  water.  Next  to  the  kidneys,  the  skin  in  man  is  the  most  important 
means  for  the  elimination  of  water.     As  the  glands  of  the  skin  and  the 

1  Zeitschr.  f.  physiol.  Chem.,  17,  25,  and  32. 

2  See  Lamois  and  Martz,  Maly's  Jahresber.,  27,  40. 

3  Aldrich,  Journ  of  Expt.  Med.,  1;  Beckmann,  Maly's  Jahresber.,  26,  566. 

4  Ber.  d.  deutsch.  chem.  Gesellsch.,  29  and  31. 

6De  Jonge,  Zeitschr.  f.  physiol.  Chem.,  3;  Zaleski,  Hoppe-Seyler's  Med.-chem. 
Untersuch.,  85;  Faust,  Arch.  f.  exp.  Path.  u.  Pharm.,  41;  Jornara  and  Casali,  Maly's 
Jahresber.,  3;  Faust,  Arch.  f.  exp.  Path.  u.  Pharm.,  47,  49;  Bertrand,  Compt.  rend., 
135;    Bertrand  and  Phisalix,  ibid. 


PERSPIRATION.  595 

kidneys  stand  near  to  each  other  in  regard  to  their  functions,  they  may  to 
a  certain  extent  act  vicariously. 

The  circumstances  which  influence  the  secretion  of  perspiration  are 
very  numerous,  and  the  quantity  of  sweat  secreted  must  consequently 
vary  considerably.  The  secretion  differs  for  different  parts  of  the  skin, 
and  it  has  been  stated  that  the  perspiration  of  the  cheek,  that  of  the  palm 
of  the  hand,  and  that  under  the  arm  stand  to  each  other  as  100:90:45. 
From  the  unequal  secretion  on  different  parts  of  the  body  it  follows  that 
no  results  as  to  the  quantity  of  secretion  for  the  entire  surface  of  the  body 
can  be  calculated  from  the  quantity  secreted  by  a  small  part  of  the  skin  in 
a  given  time.  In  determining  the  total  quantity  a  stronger  secretion  is 
as  a  rule  produced,  and  as  the  glands  can  with  difficulty  work  for  a  long 
time  with  the  same  energy,  it  is  hardly  correct  to  estimate  the  quantity 
of  secretion  per  day  from  a  strong  secretion  during  only  a  short  time. 

The  perspiration  obtained  for  investigation  is  never  quite  pure,  but 
contains  cast-off  epidermis-cells,  also  cells  and  fat-globules  from  the  seba- 
ceous glands.  Filtered  perspi ration  is  a  clear,  colorless  fluid  with  a  salty 
taste  and  of  different  odors  from  different  parts  of  the  body.  The  physio- 
logical reaction  is  acid,  according  to  most  statements.  Under  certain  con- 
ditions also  an  alkaline  sweat  may  be  secreted  (Trumpy  and  Luchsinger, 
Heuss).  An  alkaline  reaction  may  also  depend  on  a  decomposition  with 
the  formation  of  ammonia.  According  to  a  few  investigators  the  physio- 
logical reaction  is  alkaline,  and  an  acid  reaction  depends,  according  to  them, 
upon  an  admixture  of  fatty  acids  from  the  sebum.  Camerer  found 
that  the  reaction  of  human  perspiration  in  certain  cases  was  acid  and  in 
others  alkaline.  Moriggia  found  that  the  sweat  from  herbivora  was 
ordinarily  alkaline,  while  that  from  carnivora  was  generally  acid.  Accord- 
ing to  Smith  '  horse's  sweat  is  strongly  alkaline. 

The  specific  gravity  of  human  perspiration  varies  between  1.001  and 
1.010.  It  contains  977.4-995.6  p.  m.,  average  about  982  p.  m.  water.  The 
solids  are  4.4-22.6  p.  m.  The  molecular  concentration  is  also  very  variable 
and  the  freezing-point  depression  depends  essentially  upon  the  content  of 
Nad.  Ardin-Delteil  found  J  =-0.08- 0.46°,  average -0.237°.  Brieger 
and  Disselhorst  2  found  with  perspiration  containing  2.9,  7.07  and  13.5 
p.  m.  XaCl  that  the  J  was  equal  to  -0.322°,  -0.60S°and  -1.002°,  respec- 
tively. The  organic  bodies  are  neutral  fats,  cholesterin,  volatile  fatty  acids, 
traces  of  proteid  (according  to  Leclerc  and  Smith  always  in  horses,  and 


1  Trumpy  and  Luchsinger,  Pfliiger's  Arch.,  18;  Heuss,  Maly's  Jahresber.,  22; 
Camerer,  Zeitschr.  f.  Biologie,  41 ;  Moriggia,  Moleschott  's  Untersuch.  zur  Xaturlehre, 
11;  Smith,  Journ.  of  Physiol.,  11.  In  regard  to  the  older  literature  on  perspiration, 
see  Hermann's  Handbuch,  5,  Thl.  1,  421  and  543. 

'Ardin-Delteil,  Maly's  Jahresber.,  30;  Breiger  and  Disselhorst,  Deutsch.  med. 
Wochenschr.,  29. 


596  THE  SKIN  AND  ITS  SECRETIONS. 

according  to  Gaube  regularly  in  man,  while  Leube  *  claims  only  some- 
times after  hot  baths,  in  Bright's  disease,  and  after  the  use  of  pilocarpin), 
also  creatinine  (Capranica),  aromatic  oxyacids,  ethereal-sulphuric  acids  of 
phenol  and  skatoxyl  (Kast  2),  sometimes  also  of  indoxyl,  and  lastly  urea. 
The  quantity  of  urea  has  been  determined  b}r  Argutinsky.  In  two 
steam-bath  experiments,  in  which  in  the  course  of  £  and  f  hour  respectively 
he  obtained  225  and  330  c.  c.  of  perspiration,  he  found  1.61  and  1.24  p.  m. 
urea.  Of  the  total  nitrogen  of  the  perspiration  in  these  two  experiments 
68.5  per  cent  and  74.9  per  cent  respectively  belong  to  the  urea.  From 
Argutinsky 's  experiments,  and  also  from  those  of  Cramer,3  it  follows  that 
of  the  total  nitrogen  a  portion  not  to  be  disregarded  is  eliminated  by  the 
perspiration.  This  portion  was  indeed  12  per  cent  in  an  experiment  of 
Cramer  at  high  temperature  and  powerful  muscular  activity.  Cramer  has 
also  found  ammonia  in  the  perspiration.  In  uraemia,  and  in  anuria  in 
cholera,  urea  may  be  secreted  in  such  quantities  by  the  sweat-glands  that 
crystals  deposit  upon  the  skin.  The  mineral  bodies  consist  chiefly  of 
sodium  chloride  with  some  potassium  chloride,  alkali  sulphate,  and  phos- 
phate. The  relative  quantities  of  these  in  perspiration  differ  materially 
from  the  quantities  in  the  urine  (Favre,4  Kast).  The  relationship, 
according  to  Kast,  is  as  follows: 


Chlorine 

In  perspiration 1 

In  urine 1 


Phosphate 
0.0015 
0.1320 


Sulphate 
0.009 
0.397 


Kast  found  that  the  proportion  of  ethereal-sulphuric  acid  to  the  sul- 
phate-sulphuric acid  in  perspiration  was  1 :  12.  After  the  administration  of 
aromatic  substances  the  ethereal-sulphuric  acid  does  not  increase  to  the 
same  extent  in  the  perspiration  as  in  the  urine  (see  Chapter  XV). 

Sugar  may  pass  into  the  perspiration  in  diabetes,  but  the  passage  of  the  bile- 
coloring  matters  has  not  been  positively  shown  in  this  secretion.  Benzoic  acid, 
succinic  acid,  tartaric  acid,  iodine,  arsenic,  mercuric  chloride,  and  quinine  pass 
into  the  perspiration.  Uric  acid  has  also  been  found  in  the  perspiration  in  gout 
and  cystin  in  cystinuria. 

Chromhidrosis  is  the  name  given  to  the  secretion  of  colored  perspiration. 
Sometimes  perspiration  has  been  observed  to  be  colored  blue  by  indigo  (Bizio),  by 
pyocyanin,  or  by  ferro-phosphate  (Kollmann  5).  True  blood-sweat,  in  which 
blood-corpuscles  exude  from  the  opening  of  the  glands,  has  also  been  observed. 

The  exchange  of  gas  through  the  skin  in  man  Is  of  very  little  importance 
compared  with  the  exchange  of  gas  by  the  lungs.    The  absorption  of  oxy- 

1  Leclerc,  Compt.  rend.,  107;  Gaube,  Maly's  Jahresber.,  22;  Leube,  Virchow's 
Arch.,  48  and  50,  and  Arch.  f.  klin.  Med.,  7. 

2  Capranica,  Maly's  Jahresber.,  12;   Kast,  Zeitschr.  f.  physiol.  Chem.,  11. 

3  Argutinsky,  Pfliiger's  Arch.,  46;  Cramer,  Arch.  f.  Hygiene,  10. 

4  Compt.  rend.,  35,  and  Arch.  gen<Sr.  de  Med.  (5),  2. 

6  Bizio,  Wien.  Sitzungsber. ,  39;  Kollmann,  cited  from  v.  Gorup-Besanez's  Lehr- 
buch,  4.  Aufl.,  555. 


EXCHANGE  OF  GAS   THROUGH   THE  SKIN.  597 

gen  by  the  skin,  which  was  first  shown  by  Rkgxault  and  Reiset,  is  very 
small.  The  quantity  of  carbon  dioxide  eliminated  by  the  skin  increases 
With  the  rise  of  temperature  (Aubert,  Rohrig,  Fubini  and  Ronchi,  Bar- 
ratt  l).  It  is  also  greater  in  light  than  in  darkness.  It  is  greater  during 
digestion  than  when  fasting,  and  greater  after  a  vegetable  than  after  an 
animal  diet  (Fubini  and  Ronchi).  The  quantity  calculated  by  various 
investigators  for  the  entire  skin  surface  in  twenty-four  hours  varies  between 
2.23  and  32.8  grams.2  In  a  horse,  Zuntz  with  Lehmaxn  and  Hagemaxx  3 
found  for  twenty-four  hours  an  elimination  of  carbon  dioxide  by  the  skin 
and  intestine  which  amounted  to  nearly  3  per  cent  of  the  total  respiration. 
Less  than  four-fifths  of  this  carbon  dioxide  came  from  the  skin  respiration. 
According  to  the  same  investigators  the  skin  respiration  equals  2\  per  cent 
of  the  simultaneous  lung  respiration. 

1  Aubert,  Pfliiger's  Arch.,  6;  Rohrig,  Deutsch.  Klin.,  1872,  209;  Fubini  and  Ronchi, 
Moleschott's  Untersuch.  z.  Naturlehre,  12;   Barratt,  Journ.  of  Physiol.,  21. 

2  See  Hoppe-Seyler,  Physiol.  Chem.,  580. 

s  Du  Bois-Reymond 's  Arch.,  1894,  and  Maly's  Jahresber.,  24. 


CHAPTER  XVII. 

CHEMISTRY  OF  RESPIRATION. 

During  life  a  constant  exchange  of  gases  takes  place  between  the 
animal  body  and  the  surrounding  medium.  Oxygen  is  inspired  and  carbon 
dioxide  expired.  This  exchange  of  gases,  which  is  called  respiration,  is 
brought  about  in  man  and  vertebrates  by  the  nutritive  fluids,  blood  and 
lymph,  which  circulate  in  the  body  and  which  are  in  constant  communica- 
tion with  the  outer  medium  on  one  side  and  the  tissue-elements  on  the 
other.  Such  an  exchange  of  gaseous  constituents  may  take  place  wherever 
the  anatomical  conditions  offer  no  obstacle,  and  in  man  it  may  go  on  in  the 
intestinal  tract,  through  the  skin,  and  in  the  lungs.  As  compared  with 
the  exchange  of  gas  in  the  lungs,  the  exchange  already  mentioned,  which 
occurs  in  the  intestine  and  through  the  skin,  is  very  insignificant.  For  this 
reason  we  will  discuss  in  this  chapter  only  the  exchange  of  gas  between  the 
blood  and  the  air  of  the  lungs  on  one  side  and  the  blood  and  lymph  and 
the  tissues  on  the  other.  The  first  is  often  designated  as  external  respira- 
tion, and  the  other,  internal  respiration. 

I.  The  Gases  of  the  Blood. 

Since  the  pioneer  investigations  of  Magnus  and  Lothar  Meyer  the 
gases  of  the  blood  have  formed  the  subject  of  repeated  careful  investiga- 
tions bjr  prominent  experimenters,  among  whom  must  be  mentioned  first 
C.  Ludwig  and  his  pupils  and  E.  Pfluger  and  his  school.  By  these  inves- 
tigations not  only  has  science  been  enriched  by  a  mass  of  facts,  but  also 
the  methods  themselves  have  been  made  more  perfect  and  accurate.  In 
regard  to  these  methods,  as  also  in  regard  to  the  laws  of  the  absorption  of 
gases  by  liquids,  dissociation,  and  related  questions,  the  reader  is  referred 
to  text-books  on  physiology,  on  physics,  and  on  gasometric  analysis. 

The  gases  occurring  in  blood  under  physiological  conditions  are  oxygen, 
carbon  dioxide,  and  nitrogen,  and  traces  of  argon.  The  nitrogen  is  found 
only  in  very  small  quantities,  on  an  average  1.8  vols,  per  cent.  The  quan- 
tity is  here,  as  in  all  following  experiments,  calculated  for  0°  C.  and  760  mm. 
pressure.  The  nitrogen  seems  to  be  simply  absorbed  by  the  blood,  at 
least  in  great  part.  It  appears,  like  argon,  to  play  no  direct  part  in  the 
processes  of  life,  and  its  quantity  varies  but  slightly  in  the  blood  of  differ- 
ent blood-vessels. 

598 


QUANTITY  OF  OXYGEN   AND  CARBON  DIOXIDE.         ■     599 

The  oxygen  and  carbon  dioxide  behave  otherwise,  as  their  quantities 
have  significant  variations,  not  only  in  the  blood  from  different  blood- 
ls,  but  also  because  many  conditions,  such  as  a  difference  in  the  rapid- 
ity of  circulation,  a  different  temperature,  rest  and  activity,  cause  a  change. 
In  regard  to  the  gases  they  contain  the  greatest  difference  is  observable 
between  the  blood  of  the  arteries  and  that  of  the  veins. 

The  quantity  of  oxygen  in  the  arterial  blood  of  dogs  is  on  an  average 
22  vols,  per  cent  (Pfluger).  In  human  blood  Setschenow  found  about 
the  same  quantity,  namely,  21.6  vols,  per  cent.  Lower  figures  have  been 
found  for  rabbit's  and  bird's  blood,  respectively  13.2  per  cent  and  10-15 
per  cent  (Walter,  Jolyet).  Venous  blood  in  different  vascular  regions 
has  very  variable  quantities  of  oxygen.  By  summarizing  a  great  number 
of  analyses  by  different  experimenters  Zuxtz  has  calculated  that  the 
venous  blood  of  the  right  side  of  the  heart  contains  on  an  average  7.15  per 
cent  less  oxygen  than  the  arterial  blood. 

The  quantity  of  carbon  dioxide  in  the  arterial  blood  (of  dogs)  is  30  to 
40  vols,  per  cent  (Ludwig,  Setschexow,  Pfluger,  P.  Bert,  and  others), 
most  generally  about  40  per  cent.  Setschenow  found  40.3  vols,  per  cent 
in  human  arterial  blood.  The  quantity  of  carbon  dioxide  in  venous  blood 
varies  still  more  (Ludwig,  Pfluger,  and  their  pupils,  P.  Bert,  Mathieu 
and  Ubbain,  and  others).  According  to  the  calculations  of  Zuntz  the 
venous  blood  of  the  right  side  of  the  heart  contains  about  8.2  per  cent  more 
carbon  dioxide  than  the  arterial.  The  average  amount  may  be  put  down 
as  48  vols,  per  cent.  Holmgren  found  in  blood  after  asphyxiation  even 
69.21  vols,  per  cent  carbon  dioxide.1 

I  bcygen  is  absorbed  only  to  a  small  extent  by  the  plasma  or  serum,  in 
which  Pfluger  found  but  0.26  per  cent.  The  greater  part  or  nearly  all  of 
the  oxygen  is  loosely  combined  with  the  haemoglobin.  The  quantity  of 
oxygen  which  is  contained  in  the  blood  of  the  dog  corresponds  closely  to 
the  quantity  which  from  the  activity  of  the  haemoglobin  we  should  expect 
to  combine  with  oxygen,  and  from  the  quantity  of  haemoglobin  contained 
therein.  It  is  difficult  to  ascertain  how  far  the  circulating  arterial  blood 
is  saturated  with  oxygen,  as  immediately  after  bleeding  a  loss  of  oxygen 
always  takes  place.  Still  it  seems  to  be  unquestionable  that  it  is  not  quite 
completely  saturated  with  oxygen  in  life. 

The  carbon  dioxide  of  the  blood  occurs  in  part,  and  indeed,  according 
to  the  investigations  of  Alex.  Schmidt,2  Zuntz,3  and  L.  Fredericq,4  to 


1  All  the  figures  given  above  may  be  found  in  Zuntz 's  "Die  Gase  des  Blutes"  in 
Hermann's  Handbuch  d.  Physiol.,  4,  Thl.  2,  33-43,  which  also  contains  detailed  state- 
ments and  the  pertinent  literature. 

2  Ber.  d.  k.  sachs.  Gesellsch.  d.  Wissensch.,  math.-phys.  Klasse,  19,  1S67. 

3  Centralbl.  f.  d.  med.  Wissensch.,  1807,  529. 

4  Recherches  sur  la  constitution  du  Plasma  sanguin,  1878,  50,  51 


600  CHEMISTRY  OF  RESPIRATION. 

the  extent  of  at  least  one  third  in  the  blood-corpuscles,  also  in  part,  and 
in  fact  the  greatest  part,  in  the  plasma  or  serum. 

The  carbon  dioxide  of  the  red  blood-corpuscles  is  loosely  combined,  and 
the  constituent  of  these  cells  which  unites  with  the  C02  seems  to  be  the 
alkali  combined  with  phosphoric  acid,  oxyhemoglobin,  or  haemoglobin,  and 
globulin  on  one  side  and  the  haemoglobin  itself  on  the  other.  That  in  the 
red  blood-corpuscles  alkali  phosphate  occurs  in  such  quantities  that  it 
may  be  of  importance  in  the  combination  with  carbon  dioxide  is  not  to  be 
doubted;  and  it  must  be  allowed  that  from  the  diphosphate,  by  a  greater 
partial  pressure  of  the  carbon  dioxide,  monophosphate  and  alkali  carbonate 
are  formed,  while  by  a  lower  partial  pressure  of  the  carbon  dioxide  the  mass 
action  of  the  phosphoric  acid  comes  again  into  play,  so  that,  with  the  carbon 
dioxide  becoming  free,  a  re-formation  of  alkali  diphosphate  takes  place.  It 
is  generally  admitted  that  the  blood-coloring  matters,  especially  the  oxy- 
hemoglobin which  can  expel  carbon  dioxide  from  sodium  carbonate  in 
vacuo,  act  like  acids;  and  as  the  globulins  also  act  similarly  (see  below), 
these  bodies  may  also  occur  in  the  blood-corpuscles  as  an  alkali  com- 
bination. The  alkali  of  the  blood-corpuscles  must  therefore,  according  to 
the  law  of  mass  action,  be  divided  between  the  carbon  dioxide,  phos- 
phoric acid,  and  the  other  constituents  of  the  blood-corpuscles  which 
possess  acidic  properties,  and  among  these  especially  the  blood-pigments, 
because  the  globulin  can  hardly  be  of  importance  on  account  of  its  small 
quantity.  By  greater  mass  action  or  greater  partial  pressure  of  the  car- 
bon dioxide,  bicarbonate  must  be  formed  at  the  expense  of  the  diphos- 
phates and  the  other  alkali  combinations,  while  at  a  diminished  partial 
pressure  of  the  same  gas,  with  the  escape  of  carbon  dioxide,  the  alkali 
diphosphate  and  the  other  alkali  combinations  must  be  re-formed  at  the  cost 
of  the  bicarbonate. 

Haemoglobin  must  nevertheless,  as  the  investigations  of  Setschemow  * 
and  Zuntz,  and  especially  those  of  Bohr  and  Torup,2  have  shown,  be  able 
to  hold  the  carbon  dioxide  loosely  combined  even  in  the  absence  of  alkali. 
Bohr  has  also  found  that  the  dissociation  curve  of  the  carbon-dioxide 
haemoglobin  corresponds  essentially  to  the  curve  of  the  absorption  of  carbon 
dioxide,  on  which  ground  he  and  Torup  consider  the  haemoglobin  itself  as 
of  importance  in  the  binding  of  the  carbon  dioxide  of  the  blood,  and  not 
its  alkali  combinations.  According  to  Bohr  the  haemoglobin  takes  up 
the  two  gases,  oxygen  and  carbon  dioxide,  simultaneously  by  the  oxygen 
uniting  with  the  pigment  nucleus  and  the  carbon  dioxide  with  the  proteid 
component. 

The  chief  part  of  the  carbon  dioxide  of  the  blood  is  found  in  the  blood- 

1  Centralbl.  f.  d.  med.  Wissensch.,  1877.  See  also  Zuntz  in  Hermann's  Handbuch,. 
76. 

2  Zuntz,  1.  c,  76;   Bohr,  Maly's  Jahresber.,  17;  Torup,  ibid. 


THE   CARBON  DIOXIDE   OF    THE   SERUM.  601 

plasma  or  the  blood-serum,  which  follows  from  the  fact  that  the  serum  is 
richer  in  carbon  dioxide  than  the  corresponding  blood  itself.  By  experi- 
ments with  the  air-pump  on  blood-serum  it  has  been  found  that  the  chief 
pari  of  the  carbon  dioxide  contained  in  the  serum  is  given  off  in  a  vacuum, 
while  a  smaller  part  can  be  removed  only  after  the  addition  of  an  aci  !. 
The  red  blm >d-corpuscles  also  act  as  an  acid,  and  therefore  in  blood  all  the 
carbon  dioxide  is  expelled  in  vacuo.  Hence  a  part  of  the  carbon  dioxids 
is  in  firm  chemical  combination  in  the  serum. 

Absorption  experiments  with  blood-serum  have  shown  us  further  that 
the  carbon  dioxide  which  can  be  pumped  out  is  in  great  part  loosely  chem- 
ically combined,  and  from  this  loose  combination  of  the  carbon  dioxide 
it  necessarily  follows  that  the  serum  must  also  contain  simply  absorbed 
carbon  dioxide.  For  the  form  of  binding  of  the  carbon  dioxide  contained 
in  the  serum  or  the  plasma  there  are  the  three  following  possibilities:  1.  A 
part  of  the  carbon  dioxide  is  simply  absorbed;  2.  Another  part  is  in  loose 
chemical  combination;   3.  A  third  part  is  in  firm  chemical  combination. 

The  quantity  of  simply  absorbed  carbon  dioxide  has  not  been  exactly 
determined.  Setschenow  '  considers  the  quantity  in  dog-serum  to  be 
about  ^  of  the  total  quantity  of  carbon  dioxide  of  the  blood.  According 
to  the  tension  of  the  carbon  dioxide  in  the  blood  and  its  absorption  coeffi- 
cient, the  quantity  seems  to  be  still  smaller. 

The  quantity  of  carbon  dioxide  in  the  blood-serum  which  is  combined  by 
a  firm  chemical  union  depends  upon  the  quantity  of  simple  alkali  carbon- 
ate in  the  serum.  This  amount  is  not  known,  and  it  cannot  be  determined 
either  by  the  alkalinity  found  by  titration,  nor  can  it  be  calculated  from  the 
excess  of  alkali  found  in  the  ash,  because  the  alkali  is  not  only  combined  with 
carbon  dioxide,  but  also  with  other  bodies,  especially  with  proteid.  The 
quantity  of  carbon  dioxide  in  firm  chemical  combination  cannot  be  ascer- 
tained after  pumping  out  in  vacuo  without  the  addition  of  acid,  because  to 
all  appearances  certain  active  constituents  of  the  serum,  acting  like  acids, 
expel  carbon  dioxide  from  the  simple  carbonate.  The  quantity  of  carbon  diox- 
ide not  expelled  from  dog-serum  by  vacuum  alone  without  the  addition  of 
acid  amounts  to  4.9  to  9.3  vols,  per  cent,  according  to  the  determinations  of 
Pfluger.2 

From  the  occurrence  of  simple  alkali  carbonates  in  the  blood-scrum  it 
naturally  follows  that  a  part  of  the  loosely  combined  carbon  dioxide  of  the 
serum  which  can  be  pumped  out  must  exist  as  bicarbonate.  The  occur- 
rence1 of  this  combination  in  the  blood-serum  has  also  been  directly  shown. 
In  experiments  with  the  pump,  as  well  as  in  absorption  experiments,  the 
serum  behaves  in  other  ways  different  from  a  solution  of  bicarbonate,  or 
carbonate  of  a  corresponding  concentration;   and  the  behavior  of  the  loosely 

1  CentralM.  f.  d.  med.  Wissensch.,  1877. 

2  E.  Pfliiger,  Ueber  die  Kohlensaure  des  Blutes,  Bonn,  1SG4.  11.  Cited  from 
Zuntz  in  Hermann's  Handbuch,  65. 


602  CHEMISTRY  OF- RESPIRATION. 

combined  carbon  dioxide  in  the  serum  can  be  explained  only  by  the  occur- 
rence of  bicarbonate  in  the  serum.  By  means  of  vacuum  the  serum  always 
allows  much  more  than  one  half  of  the  carbon  dioxide  to  be  expelled,  and 
it  follows  from  this  that  in  the  pumping  out  not  only  may  a  dissociation  of 
the  bicarbonate  take  place,  but  also  a  conversion  of  the  double  sodium 
carbonate  into  a  simple  salt.  As  we  know  of  no  other  carbon-dioxide  com- 
bination besides  the  bicarbonate  in  the  serum  from  which  the  carbon 
dioxide  can  be  set  free  by  simple  dissociation  in  vacuo,  it  must  be 
assumed  that  the  serum  contains  other  weak  acids,  in  addition  to  the 
carbon  dioxide,  which  contend  with  it  for  the  alkalies,  and  which  expel  the 
carbon  dioxide  from  simple  carbonates  in  vacuo.  The  carbon  dioxide 
which  is  expelled  by  means  of  the  pump  and  which,  without  regard  to  the 
quantity  merely  absorbed,  is  generally  designated  as  ' '  carbon  dioxide  in 
loose  chemical  combination, ' '  is  thus  only  obtained  in  part  in  dissociable 
loose  combinations;  in  part  it  originates  from  the  simple  carbonates,  from 
which  it  is  expelled  in  vacuo  by  other  weak  acids. 

These  weak  acids  are  thought  to  be  in  part  phosphoric  acid  and  in  part 
globulins.  The  importance  of  the  alkali  phosphates  for  the  carbon-dioxide 
combination  has  been  shown  by  the  investigations  of  Fernet;  but  the 
quantity  of  these  salts  in  the  serum  is,  at  least  in  certain  kinds  of  blood, 
for  example  in  ox-serum,  so  small  that  it  can  hardly  be  of  importance.  In 
regard  to  the  globulins  Setschenow  is  of  the  opinion  that  they  do  not  act 
as  acids  themselves,  but  form  a  combination  with  carbon  dioxide,  produc- 
ing carboglobulinic  acid,  which  unites  with  the  alkali.  According  to  Ser- 
toli,1 whose  views  have  found  a  supporter  in  Torup,  the  globulins  them- 
selves are  the  acids  which  are  combined  with  the  alkali  of  the  blood-serum. 
In  both  cases  the  globulins  would  form,  directly  or  indirectly,  that  chief 
constituent  of  the  plasma  or  of  the  blood-serum  which,  according  to  the  law 
of  mass  action,  contends  with  the  carbon  dioxide  for  the  alkalies.  By  a 
greater  partial  pressure  of  the  carbon  dioxide  the  latter  deprives  the  globu- 
lin alkali  of  a  part  of  its  alkali  and  bicarbonate  is  formed;  by  low  partial 
pressure  the  carbon  dioxide  escapes  and  the  bicarbonate  is  abstracted  by 
the  globulin  alkali. 

In  the  foregoing  it  has  been  assumed  that  the  alkali  is  the  most  essential 
and  important  constituent  of  the  blood-serum,  as  well  as  of  the  blood  in 
general,  in  uniting  with  the  carbon  dioxide.  The  fact  that  the  quantity  of 
carbon  dioxide  in  the  blood  greatly  diminishes  with  a  decrease  in  the  quan- 
tity of  alkali  strengthens  this  assumption.  Such  a  condition  is  found,  for 
example,  after  poisoning  with  mineral  acids.  Thus  Walter  found  only 
2-3  vols,  per  cent  carbon  dioxide  in  the  blood  of  rabbits  into  whose  stomachs 
hydrochloric  acid  had  been  introduced.  In  the  comatose  state  of  diabetes 
mellitus  the  alkali  of  the  blood  seems  to  be  in  great  part  saturated  with  acid 

1  Hoppe-Seyler,  Med.  chem.  Untersuch.  • 


GASES  OF  THE  LYMPH,  ETC.  603 

combinations,  .?-oxybutyrie  acid  (St.\i>klman\,. Minkowski), and  Minkowski1 
found  only  ;>.3  vols,  per  cent  carbon  dioxide  in  the  blood  in  diabetic  coma. 

Gases  of  the  Lymph  and  Secretions. 

The  gases  of  the  lymph  are  the  same  as  in  the  blood-serum,  and  the 
lymph  stands  close  to  the  blood-serum  in  regard  to  the  quantity  of  the 
various  gases,  as  well  as  to  the  kind  of  carbon-dioxide  combination.  The 
investigations  of  Daenhardt  and  Hensen3  on  the  gases  of  human  lymph 
are  at  hand,  but  it  still  remains  a  question  whether  the  lymph  investigated 
was  quite  normal.  The  gases  of  normal  dog-lymph  were  first  investigated 
by  Hammarsten.8  These  gases  contained  traces  of  oxygen  and  consisted 
of  •ST. 4-53.1  per  cent  CO,  and  1.6  per  cent  N  at  0°  C.  and  TOO  mm.  Hg  pres- 
sure. About  one  half  of  the  carbon  dioxide  was  in  firm  chemical  com- 
bination. The  quantity  was  greater  than  in  the  serum  from  arterial 
blood,  but  smaller  than  from  venous  blood. 

The  remarkable  observation  of  Buchner  that  the  lymph  collected  after 

asphyxiation  is  poorer  in  carbon  dioxide  than  that  of  the  breathing  animal 

is  explained  by  ZuNTZ  i  by  the  formation  of  acid  immediately  after  death  in 

the  tissues,  and  especially  in  the  lymphatic  glands,  and  this  acid  decom- 

-  the  alkali  carbonates  of  the  lymph  in  part. 

The  secretions  with  the  exception  of  the  saliva,  in  which  Pflugeb  and 
Kulz  found  respectively  0.6  per  cent  and  1  per  cent  oxygen,  are  nearly 
free  from  oxygen.  The  quantity  of  nitrogen  is  the  same  as  in  blood,  and 
the  chief  mass  of  the  gases  consists  of  carbon  dioxide.  The  quantity  of 
this  gas  is  chiefly  dependent  upon  the  reaction,  i.e.,  upon  the  quantity  of 
alkali.  This  follows  from  the  analyses  of  Pfluger.  He  found  19  per  cent 
carbon  dioxide  removable  by  the  air-pump  and  54  per  cent  firmly  com- 
bined carbon  dioxide  in  a  strongly  alkaline  bile,  but,  on  the  contrary, 
6.6  per  cent  carbon  dioxide  removable  by  the  air-pump  and  0.S  per  cent 
firmly  combined  carbon  dioxide  in  a  neutral  bile.  Alkaline  saliva  is  also 
very  rich  in  carbon  dioxide.  As  average  for  two  analyses  made  by  Pflu- 
ger  of  the  submaxillary  saliva  of  a  dog  we  have  27.5  per  cent  carbon  diox- 
ide removable  by  the  air-pump  and  47.4  per  cent  chemically  combined 
carbon  dioxide,  making  a  total  of  74.9  percent.  Kulz  5  found  a  maxi- 
mum of  65.78  per  cent  carbon  dioxide  for  the  parotid  saliva,  of  which 
3.31  per  cent  was  removable  by  the  air-pump  and  62.47  per  cent  was 
firmly  combined.     From  these  and  other  statements  on  the  quantity  of 

'Walter,  Arch.  f.  cxp.  Path.  u.  Pharm.,  7;  Stadelmann,  ibid.,  17;  Minkowski, 
Mittheil.  a.  <1  med.  Klink  in  Konigsberg,  1S88. 

;  Yirchow's  Arch.,  37. 

3  Ber.  d.  k.  sachs.  Gesellsch.  d.  Wissensch.,  math.-phys.  Klasse,  23. 

1  Buchner,  Arbeiten  aus  der  physiol.  Anstalt  ni  Leipzig,  1S70;   Zuntz,  1.  c,  S5. 

5  Pfluger,  Pfluger 's  Arch.,  1  and  2;  Kulz,  Zeitschr.  f.  Biologic.  23.  It  seems 
as  if  Kitlz's  results  were  not  calculated  at  700  milligrams  Hg,  but  rather  at  1  milligram. 


604  CHEMISTRY  OF  RESPIRATION. 

carbon  dioxide  removable  by  the  air-pump  and  chemically  combined  in 
the  alkaline  secretions  it  follows  that  bodies  occur  in  them,  although  not 
an  appreciable  quantities,  which  are  analogous  to  the  proteid  bodies  of 
the  blood-serum  and  which  act  like  weak  acids. 

The  acid  or  at  any  rate  non-alkaline  secretions,  urine  and  milk,  contain, 
<on  the  contrary,  considerably  less  carbon  dioxide,  which  is  nearly  all  remov- 
able by  the  air-pump,  and  a  part  seems  to  be  loosely  combined  with  the 
sodium  phosphate.  The  figures  found  by  Pfluger  for  the  total  quantity 
■of  carbon  dioxide  in  milk  and  urine  are  10  and  18.1-19.7  per  cent  respec- 
tively. 

Ewald  x  has  made  investigations  on  the  quantity  of  gas  in  pathological 
transudates.  He  found  only  traces,  or  at  least  only  very  insignificant 
quantities  of  oxygen  in  these  fluids.  The  quantity  of  nitrogen  was  about 
the  same  as  in  blood ;  that  of  carbon  dioxide  was  greater  than  in  the  lymph 
(of  dogs),  and  in  certain  cases  even  greater  than  in  the  blood  after  asphyxi- 
ation (dog's  blood).  The  tension  of  the  carbon  dioxide  was  greater  than 
in  venous  blood.  In  exudates  the  quantity  of  carbon  dioxide,  especially 
that  firmly  combined,  increases  with  the  age  of  the  fluid,  while,  on  the 
ccontrary,  the  total  quantity  of  carbon  dioxide,  and  especially  the  quantity 
firmly  combined,  decreases  with  the  quantity  of  pus-corpuscles. 

II.   The  Exchange  of  Gas  hetween  the  Blood  on  the  One  Hand  and 
Pulmonary  Air  and  the  Tissues  on  the  Other. 

In  the  introduction  (Chapter  I,  p.  3)  it  was  stated  that  we  are  to-day  of 
the  opinion,  derived  especially  from  the  researches  of  Pfluger  and  his 
pupils,  that  the  oxidations  of  the  animal  body  do  not  take  place  in  the 
fluids  and  juices,  but  are  connected  with  the  form-elements  and  tissues.  It 
is  nevertheless  true  that  oxidations  take  place  in  the  blood,  although  only 
to  a  slight  extent;  but  these  oxidations  depend,  it  seems,  upon  the  form- 
elements  of  the  blood,  hence  it  does  not  contradict  the  above  statement 
that  the  oxidations  occur  exclusively  in  the  cells  and  chiefly  in  the  tissues. 

The  gaseous  exchange  in  the  tissues,  which  has  been  designated  internal 
respiration,  consists  chiefly  in  that  the  oxygen  passes  from  the  blood  in  the 
capillaries  to  the  tissues,  while  the  great  bulk  of  the  carbon  dioxide  of  the 
tissues  originates  therein  and  passes  into  the  blood  of  the  capillaries.  The 
exchange  of  gas  in  the  lungs,  which  is  called  external  respiration,  consists, 
as  is  seen  by  a  comparison  of  the  inspired  and  expired  air,  in  the  blood 
taking  oxygen  from  the  air  in  the  lungs  and  giving  off  carbon  dioxide. 
This  does  not  exclude  the  fact  that  in  the  lungs,  as  in  every  other  tissue,  an 
internal  respiration  takes  place,  namely,  a  combustion  with  a  consump- 
tion of  oxygen  and  formation  of  carbon  dioxide.     According  to  Bohr  and 


1  C.  A.  Ewald,  Arch.  f.  (Anat.  u.)  Physiol.,  1873  and  1876. 


DISSOCIATION  OF  OXYHEMOGLOBIN.  605 

Henriques  '  the  lungs  indeed  play  so  large  a  part  in  the  total  metabolism 
that  it  may  amount  to  6S  per  cent  of  the  same. 

What  kind  of  processes  take  part  in  this  double  exchange  of  gas?     Is 
the  gaseous  exchange  simply  the  result  of  an  unequal  tension  of  the  blood 
on  one  side  and  the  air  in  the  lungs  or  tissues  on  the  other?     Do  the  : 
pass  from  a  place  of  higher  pressure  to  one  of  a  lower,  according  to  the  laws 
of  diffusion,  or  are  other  forces  and  processes  active? 

These  questions  are  closely  related  to  that  of  the  tension  of  the  oxygen 
and  carbon  dioxide  in  the  blood  and  in  the  air  of  the  lungs  and  tissues. 

Oxygen  occurs  in  the  blood  in  a  disproportionately  large  part  as  oxy- 
hemoglobin, and  the  law  of  the  dissociation  of  oxyhemoglobin  is  of  funda- 
mental importance  in  the  study  of  the  tension  of  the  oxygen  in  the  blood. 

If  it  is  recalled  that,  according  to  Bohr,  what  is  generally  termed  oxyhaemo- 
glohin  is  a  mixture  of  haemoglobins,  which  for  one  and  the  same  oxygen  pres- 
vsure  can  unite  with  different  quantities  of  oxygen,  and  also,  as  shown  by  Sieg- 
fried, that  there  exists,  besides  the  oxyhemoglobin,  another  dissociable  oxygen 
combination  of  haemoglobin,  namely,  pseudohaemoglobin,  it  seems  that  there  are 
several  important  preliminary  questions  to  solve  before  we  come  to  a  discussion 
of  the  dissociation  conditions  of  oxyhemoglobin.  As  the  above  statements  are 
in  part  contradicted  and  in  part  not  sufficiently  proved,  and  as  also,  according 
to  Hufner,  no  difference  exists  between  an  oxy haemoglobin  solution  and  a  solu- 
tion of  blood-corpuscles  in  regard  to  its  delivery  of  oxygen,  the  above  statements 
can  be  set  aside  for  the  present  and  only  the  generally  accepted  and  authori- 
tative assertions  discussed. 

For  the  understanding  of  the  laws  by  which  the  oxygen  is  taken  up 
by  the  blood  in  the  alveoli  of  the  lungs  the  investigations  on  the  dissocia- 
tion of  oxyhemoglobin  are  important,  and  those  especially  which  relate  to 
the  dissociation  at  the  temperature  of  the  body  are  of  great  physiolog- 
ical int  rest.  Several  investigators  have  experimented  on  this  subject, 
especially  G.  Hufner.2  He  has  proved  the  important  fact  that  a 
freshly  prepared  solution  of  pure  oxyhemoglobin  crystals  does  not  act 
unlike  freshly  defibrinated  blood  as  regards  the  dissociation  of  oxyhemo- 
globin. He  also  showed  that  the  dissociation  is  dependent  upon  the  con- 
centration, namely,  that  at  a  given  pressure  a  dilute  solution  is  more 
strongly  dissociated  than  a  more  concentrated  solution.  He  found  for 
solutions  containing  14  per  cent  hemoglobin  that  the  dissociation  at 
35°  C.  and  an  oxygen  partial  pressure  of  75  mm.  Hg  was  only  very  insig- 
nificant and  only  little  greater  than  with  a  partial  pressure  of  152  mm. 
In  the  first  instance  96.89  per  cent  of  the  total  pigment  was  presenl 
a<  oxyhemoglobin  and  3.11  per  cent  as  hemoglobin,  while  in  the  other 
case,  at  152  mm.  pressure,  the  respective  figures  were  9S.42  per  cent  and 
1.5S  per  cent.  The  dissociation  becomes  stronger  first  with  an  oxvgcn 
partial  pressure  of   about  75  mm.   Hg  and  downwards  and   corresponds 

1  Centralbl.  f.  Physiol.,  6,  and  Maly's  Jahresber.,  27. 

2  Du  Bois-Reymond's  Arch.,  1S90,  where  the  older  works  on  this  topic  are  cited. 


606  CHEMISTRY  OF  RESPIRATION. 

to  an  increase  in  the  quantity  of  reduced  haemoglobin;  but  even  with  an 
oxygen  partial  pressure  of  50  mm.  Hg  the  quantity  of  haemoglobin  was. 
only  4.6  per  cent  of  the  total  pigment. 

From  these  and  older  researches  by  Hufner,1  which  were  made  at 
35°  or  39°  C,  it  follows  that  the  partial  pressure  of  the  oxygen  may  be 
reduced  to  one  half  of  the  atmospheric  air  without  influencing  essentially 
the  quantity  of  oxygen  in  the  blood  or  a  corresponding  solution  of  oxy- 
haemoglobin.  This  corresponds  well  with  the  experience  of  Frankel  and 
Geppert  2  on  the  action  of  diminished  air-pressure  on  the  quantity  of 
oxygen  in  the  blood  in  dogs.  With  an  air-pressure  of  410  mm.  Hg  they 
found  the  quantity  of  ox}^gen  in  arterial  blood  to  be  normal.  With  a. 
pressure  of  378-365  mm.  it  was  slightly  diminished,  and  only  on  decreas- 
ing the  pressure  to  300  mm.  was  the  diminution  considerable.  The  lowest 
limit  for  the  oxygen  pressure  in  the  alveoli  air  at  which  the  normal  qualita- 
tive and  quantitative  exchange  of  material  may  go  on  has  been  found  by 
A.  Loewy  3  to  be  equal  to  a  pressure  of  30  mm.  Hg.  The  reason  why  on 
lowering  the  alveolar  oxygen  tension  below  this  limit  the  metabolism  ap- 
pears similar  to  tissue  dyspnoea  he  explains  by  the  fact  of  such  a  marked 
increase  in  the  dissociation  of  the  oxjdiaemoglobin  that  an  insufficient  quan- 
tity of  oxygen  is  supplied  to  the  tissues.  This  opinion  is  disproved  by  the 
researches  of  Hufner  on  the  dissociation  of  oxyhaemoglobin  in  which  with 
an  oxygen  partial  pressure  of  30  mm.  Hg  about  92  per  cent  is  still  saturated 
with  oxygen.  For  this  reason  Loewy  has  made  newer  experiments  on 
the  dissociation  of  oxyhaemoglobin  in  human  blood  and  has  obtained  dif- 
ferent results  than  Hufner.  With  an  oxygen  pressure  of  36-37  mm.  Hg 
he  never  found  above  80  per  cent  saturation.  With  a  pressure  of  35  mm. 
the  saturation  was  about  77  per  cent  (Hufner  93  per  cent) ;  at  30  mm. 
75  per  cent  (Hufner  92  per  cent) ;  at  25  mm.  65  per  cent  (Hufner  about 
91  per  cent),  and  at  22-23  mm.  about  58  per  cent.  As  explanation  for 
these  differences  in  the  results  of  the  two  experimenters  Loewy  suggests 
the  possibility  that  there  possibly  exists  a  difference  in  the  combining 
power  for  oxygen  between  crystalline  haemoglobin  and  the  haemoglobin 
of  the  fresh  blood.  Still  it  must  be  remarked,  as  above  stated,  that  accord- 
ing to  the  special  investigations  of  Hufner  a  freshly  prepared  solution 
of  oxyhaemoglobin  crystals  does  not  behave  different  in  any  way  in  regard 
to  the  dissociation  of  oxyhaemoglobin  from  fresh,  defibrinated  blood.  The 
above  differences  cannot  be  satisfactorily  explained. 

The  taking  up  of  oxygen  from  the  air  is,  according  to  Rosenthal,4 

1  Du  Bois-Reymond 's  Arch.,  1890. 

2  "Ueber  die  Wirkungen  der  verdiinnten  Luft  auf  den  Organismus. "     Berlin,  1883. 

3  A.  Loewy,  "Untersuch.  liber  die  Respiration  und  Circulation,"  etc.  Berlin,. 
1895;  also  Centrarbl.  f.  Physiol.,  13,  449,  and  Arch.  f.  (Anat.  u.)  Physiol.,  1900. 

'Arch.  f.  (Anat.  u.)  Physiol.,  1898,  and  especially  1902.  See  also  Durig,  ibid., 
1903,  Suppl. 


COMPOSITION  OF  THE  ALVEOLAR  AIR.  607 

not  as  independent  upon  the  quantity  of  oxygen  as  is  generally  considered, 
based  upon  the  investigations  of  Regnault  and  Rieset.    Rosenthal  has 

found  that  at  least  with  quick  exchange  of  the  oxygen  content  of  the  air, 
the  taking  up  5f  oxygen  with  diminished  oxygen  content  diminishes  and 

with  increased  content  it  is  raised.  As  the  carbon-dioxide  excretion  is 
not  hereby  correspondingly  changed  the  oxygen  is  stored  up  in  the  tissues, 
according  to  ROSENTHAL,  when  an  increased  absorption  of  oxygen  takes 
place,  while  with  diminished  oxygen  the  deficit  is  replaced  from  the  reserve 
en  supply  of  the  tissues.  Every  cell  contains  bodies  which  fix  oxygen, 
and  when  necessary  they  readily  give  this  off.  This  oxygen  has  been 
called   intracellular  oxygen  by   ROSENTHAL. 

It  may  be  concluded  from  the  large  quantity  of  oxygen  or  oxyhemo- 
globin in  the  arterial  blood  that  the  tension  of  the  oxygen  in  the  arterial 
blood  must  be  relatively  higher.  From  the  investigations  of  several  experi- 
menters, such  as  P.  Bert,  Herter,1  and  Hupner,  who  experimented 
partly  on  living  animals  and  partly  with  haemoglobin  solutions,  we  may 
assume  the  tension  of  the  oxygen  in  arterial  blood  at  the  temperature  of 
the  body  to  be  equal  to  a  partial  oxygen  pressure  of  75-80  mm.  Hg. 

Let  us  now  compare  these  figures  with  the  tension  of  the  oxygen  in 
the  air  of  the  lungs. 

Numerous  investigations  as  to  the  composition  of  the  inspired  atmos- 
pheric air  as  well  as  the  expired  air  are  at  hand,  and  it  can  be  said  that 
these  two  kinds  of  air  at  0°  C.  and  a  pressure  of  760  mm.  Hg  have  the  fol- 
lowing average  composition  in  volume  per  cent: 

Oxygen.  Nitrogen.     Carbon  Dioxide. 

Atmospheric  air 20. 9G  79.02  0.03 

Expiredair 1G.03  79.59  I  .:;s 

The  partial  pressure  of  the  oxygen  of  the  atmospheric  air  corresponds 
at  a  normal  barometric  pressure  of  760  mm.  to  a  pressure  of  160  mm.  Hg. 
The  loss  of  oxygen  which  the  inspired  air  suffers  in  respiration  amounts  to 
about  4.93  per  cent,  while  the  expired  air  contains  about  one  hundred 
times  as  much  carbon  dioxide  as  the  inspired  air. 

The  expired  air  is  therefore  a  mixture  of  alveolar  air  with  the  residue 
of  inspired  air  remaining  in  the  air-passages;  hence  in  the  study  of  the 
gaseous  exchange  in  the  lungs  the  alveolar  air  must  first  be  considered. 
There  does  not  exist  any  direct  determination  of  the  composition  of  the 
alveolar  air  in  man,  but  only  approximate  calculations.  From  the  aver- 
age results  found  by  ViERORDT  in  normal  respiration  for  the  carbon  diox- 
ide in  the  expired  air.  4.63  per  cent.  Zuntz  2  has  calculated  the  probable 
quantity  of  carbon  dioxide  in  the  alveolar  air  as  equal  to  5.44  per  cent. 
If  we  start  from  this  value  with  the  assumption  that  the  quantity  of  nitro- 

1  Bert,  La  pression  barometrique,  Paris,  1S7S;  Herter,  Zeitschr.  f.  physiol.  Chem.,  3. 
*  See  Zuntz,  1.  c,  105  and  106. 


60S  CHEMISTRY  OF  RESPIRATION. 

gen  in  the  alveolar  air  does  not  essentially  differ  from  the  expired  air, 
and  admit  that  the  quantity  of  oxygen  in  the  alveolar  air  is  6  per  cent 
less  than  the  inspired  air,  it  will  be  seen  that  the  alveolar  air  contains  15 
per  cent  oxygen,  corresponding  to  a  partial  pressure  of  115  mm.  Hg. 

There  are  several  direct  determinations  of  the  alveolar  air  of  dogs  by 
Pfluger  and  his  pupils  Wolffberg  and  Nussbaum.1  These  determina- 
tions which  show  that  the  alveolar  air  is  not  much  richer  in  carbon  dioxide 
than  the  expired  air  have  been  performed  by  means  of  the  so-called  lung- 
catheter. 

The  principle  of  this  method  is  as  follows:  By  the  introduction  of  a  catheter 
of  a  special  construction  into  a  branch  of  a  bronchus  the  corresponding  lobe  of 
the  lung  may  be  hermetically  sealed,  -while  in  the  other  lobes  of  the  same  lung,  and 
in  the  other  lung,  the  ventilation  remains  unchanged,  so  that  no  accumulation 
of  carbon  dioxide  takes  place  in  the  blood.  When  the  cutting  off  lasts  so  long  that 
a  complete  equalization  between  the  gases  of  the  blood  and  the  retained  air  of 
the  lungs  is  assumed,  a  sample  of  this  air  of  the  lungs  is  removed  by  means  of 
the  catheter  and  analyzed. 

In  the  air  thus  obtained  from  the  lungs  Wolffberg  and  Nussbaum 
found  an  average  of  3.6  per  cent  C02.  Nussbaum  has  also  determined  the 
carbon-dioxide  tension  in  the  blood  from  the  right  heart  in  a  case  simul- 
taneous with  the  catheterization  of  the  lungs.  He  found  nearly  identical 
results,  namely,  a  carbon-dioxide  tension  of  3.84  per  cent  and  3.81  per 
cent  of  an  atmosphere,  which  also  shows  that  complete  equalization  between 
the  gases  of  the  blood  and  lungs  in  the  enclosed  parts  of  the  lungs  had 
taken  place.  From  these  investigations  it  can  be  calculated  that  the 
quantity  of  oxygen  in  the  alveolar  air  of  dogs  is  about  16  per  cent,  which 
corresponds  to  an  oxygen  partial  pressure  of  about  122  nun.  Hg.  This 
pressure  is  considerably  higher  than  the  oxygen  tension  in  arterial  blood, 
and  the  oxygen  absorption  from  the  air  of  the  lungs  takes  place  simply 
according  to  the  laws  of  diffusion. 

According  to  Bohr  2  the  facts  are  otherwise,  and  the  lungs  are  active  in 
the  taking  up  of  oxygen. 

He  experimented  on  dogs,  allowing  the  blood,  whose  coagulation  had  been 
prevented  by  the  injection  of  peptone  solution  or  infusion  of  the  leech,  to  flow 
from  one  bisected  carotid  to  the  other,  or  from  the  femoral  artery  to  the  femoral 
vein,  through  an  apparatus  called  by  him  an  hscmataerometer.  The  apparatus, 
which  is  a  modification  of  Ludwig's  rheometer  (strovmhr) ,  allowed,  according 
t«,  Bohb,  of  a  complete  interchange  between  the  gases  of  the  blood  circulating 
through  the  apparatus  and  a  quantity  of  gas  whose  composition  was  known 
at  the  beginning  of  the  experiment  and  enclosed  in  the  apparatus.  The  mixture 
of  gases  was  analyzed  after  an  equalization  of  the  gases  by  diffusion.  In  this 
way  the  tension  of  the  oxygen  and  carbon  dioxide  in  the  circulating  arterial 
blood  was  determined.  During  the  experiment  the  composition  of  the  inspired 
and  expired  air  was  also  determined,  the  number  of  inspirations  noted,  and  the 

'Wolffberg,  Pfluger's  Arch.,  6;   Nussbaum,  ibid.,  7. 
2Skand.  Arch.  f.  Physiol..  2. 


OXYGEN  TENSION  IN  THE  BLOOD.  609 

extent  of  respiratory  exchange  of  gas  measured.  To  be  able  to  make  a  comparison 
between  the  gas  tension  in  the  blood  and  in  an  expired  air  whose  composition  was 
closer  to  the  unknown  composition  of  the  alveolar  air  than  the  ordinary  expired  air, 
the  composition  of  the  expired  air  at  the  moment  it  passed  the  bifurcation  of  t he 
trachea  was  ascertained  by  special  calculation.  The  tension  of  the  gases  in  this 
"bifurcated  air"  could  be  compared  with  the  tension  of  the  gases  of  the  blood, 
and  in  such  a  way  that  the  comparison  took  place  simultaneously. 

Bohr  found  remarkably  high  results  for  the  oxygen  tension  in  arterial 
blood  in  this  series  of  experiments.  They  varied  between  101  and  144 
mm.  Hg  pressure.  In  eight  out  of  nine  experiments  on  the  breathing  of 
atmospheric  air,  and  in  four  out  of  five  experiments  on  breathing  air 
containing  carbon  dioxide,  the  oxygen  tension  in  the  arterial  blood  was 
higher  than  the  "bifurcated  air."  The  greatest  difference,  where  the  oxy- 
tension  wras  higher  in  the  blood  than  in  the  air  of  the  lungs,  was  38  mm.  Hg. 

According  to  Bohr  we  cannot  simply  explain  the  taking  up  of  oxygen 
by  the  blood  from  the  air  of  the  lungs  by  a  higher  partial  pressure  of  the 
oxygen.  The  difference  in  tension  between  the  two  sides  of  the  walls  of 
the  alveoli  therefore  may  not  be  the  only  force  which  serves  in  the  migra- 
tion of  the  oxygen  through  the  lung  tissue,  and  the  lungs  themselves  must 
exercise  an  unknown  specific  action  in  the  taking  up  of  oxygen. 

HuFNER  and  Fredericq  *  have  made  the  objection  to  Bohr's  experi- 
ments and  views  that  a  perfect  equilibrium  had  probably  not  been  attained 
between  the  air  in  the  apparatus  and  the  gases  of  the  blood.  Fredericq, 
by  new  experiments,  has  presented  strong  objections  to  the  acceptance  of 
Bohr's  findings.  On  the  other  hand  Haldane  and  Smith's2  recent 
experiments  upon  an  entirely  different  principle  show  results  which  con- 
tradict the  ordinary  doctrine  of  the  oxygen  absorption  in  the  lungs. 

Haldane 's  method  is  as  follows:  The  individual  experimented  upon  is  allowed 
to  inspire  air  containing  an  exactly  known  but  small  quantity  of  carbon  monoxide 
(0.045-0.06  per  cent),  until  no  further  absorption  of  carbon  monoxide  takes  place 
and  the  percentage  saturation  of  the  haemoglobin  in  the  arterial  blood  with 
carbon  monoxide  has  become  constant,  as  shown  by  a  special  titration  method. 
This  percentage  saturation  is  dependent  upon  the  relation  between  the  tension 
of  the  oxygen  in  the  blood  and  the  tension  of  the  carbon  monoxide,  as  known 
from  the  composition  of  the  inspired  air.  When  this  last  and  the  percentage 
saturation  with  carbon  monoxide  and  oxygen  are  known  the  oxygen  tension  in 
the  blood  can  be  easily  calculated. 

Haldaxe  and  Smith  calculate  the  tension  of  the  oxygen  in  arterial 
human  blood  at  an  average  of  26.2  per  cent  of  an  atmosphere,  i.e.,  equal 
approximately  to  200  mm.  Hg.  In  agreement  with  Bohr  the  view  is  held 
that  diffusion  alone  cannot  explain  the  passage  of  oxygen  from  the  lungs  t*> 
the  blood,  and  that  this  question  requires  further  investigation. 

1  Hiifner,  Du  Bois-Reymond's  Arch.,  1890;  Fredericq,  Centralbl.  f.  Physiol.,  7, 
and  Travaux  du  laboratoire  de  1'institut  de  physiologie  de  Liege,  5,  1896. 

2  Haldane,  Journ.  of  Physiol.,  IS;   Haldane  and  Smith,  ibid.,  20. 


610  CHEMISTRY  OF  RESPIRATION. 

As  the  haemoglobin  obtained  from  different  blood  portions  does  Dot.  according 
to  Bohr,  always  take  up  the  same  quantity  of  oxygen  for  each  gram,  so  the 
haemoglobin  within  the  blood-corpuscle  may  show  a  similar  behavior.  He  calls 
the  quantity  of  oxygen  (measured  at  0°  C.  and  760  mm.  Hg)  which  is  taken  up 
by  1  gram  of  haemoglobin  of  the  blood  at  15°  C.  and  an  oxygen  pressure  of  150  mm. 
the  specific  oxygen  capacity.1  This  quantity,  he  claims,  may  be  different  not  only 
in  different  indiviuals,  but  also  in  the  different  vascular  systems  of  the  same 
animal,  and  it  may  also  be  changed  experimentally  by  bleeding,  breathing  air 
deficient  in  oxygen,  or  poisoning.  It  is  now  evident  that  one  and  the  same  quan- 
tity of  oxygen  in  the  blood,  other  things  being  equal,  must  have  a  different  ten- 
sion according  as  the  specific  oxygen  capacity  is  greater  or  smaller.  The  tension 
of  the  oxygen,  Bohr  says,  may  be  changed  without  changing  the  quantity  of 
oxygen,  and  the  animal  body  must,  according  to  him,  have  means  of  varying  the 
tension  of  the  oxygen  in  the  tissues  in  a  short  time  without  changing  the  quantity 
of  oxygen  contained  in  the  blood.  The  great  importance  of  such  a  property  of 
the  tissues  for  respiration  is  evident;  but  it  is  perhaps  too  early  to  give  a  positive 
opinion  on  Bohr  's  statements  and  experiments. 

The  tension  of  the  carbon  dioxide  in  the  blood  has  been  determined  in 
different  ways  by  Pfluger  and  his  pupils,  Wolffberg,  Strassburg,  and 

NUSSBAUM.2 

According  to  the  aerotonometric  method  the  blood  is  allowed  to  flow  directly 
from  the  artery  or  vein  through  a  glass  tube  which  contains  a  gas  mixture  of  a 
known  composition.  If  the  tension  of  the  carbon  dioxide  in  the  blood  is  greater 
than  the  gas  mixture,  then  the  blood  gives  up  carbon  dioxide,  while  in  the  reverse 
case  it  takes  up  carbon  dioxide  from  the  gas  mixture.  The  analysis  of  the  gas 
mixture  after  passing  the  blood  through  it  will  al?o  decide  if  the  tension  of  the 
carbon  dioxide  in  the  blood  is  greater  or  less  than  in  the  gas  mixture;  and  by  a 
sufficiently  great  number  of  determinations,  especially  when  the  quantity  of  carbon 
dioxide  of  the  gas  mixture  corresponds  as  nearly  as  possible  in  the  beginning  to 
the  probable  tension  of  this  gas  in  the  blood,  we  may  learn  the  tension  of  the 
carbon  dioxide  in  the  blood. 

According  to  this  method  the  carbon-dioxide  tension  of  the  arterial 
blood  is  on  an  average  2.8  per  cent  of  an  atmosphere,  corresponding  to  a 
pressure  of  21  mm.  mercury  (Strassburg).  In  the  blood  from  the  pul- 
monary alveoli  Nussbaum  found  a  carbon-dioxide  tension  of  3.81  per  cent 
of  an  atmosphere,  corresponding  to  a  pressure  of  28.95  mm.  mercury. 
Strassburg,  who  experimented  in  non-tracheotomized  dogs  in  which  the 
ventilation  of  the  lungs  was  less  active  and  therefore  the  carbon  dioxide 
was  removed  from  the  blood  with  less  readiness,  found  in  the  venous  blood 
of  the  heart  a  carbon-dioxide  tension  of  5.4  per  cent  of  an  atmosphere, 
(  orresponding  to  a  partial  pressure  of  41.01  mm.  mercury. 

Another  method  is  the  catheterization  of  a  lobe  of  the  lungs  (ree  page 
608).  In  the  air  thus  obtained  from  the  lungs  Nussbaum  and  Wolffberg 
found  an  average  of  3.6  per  cent  C02.  Nussbaum,  as  previously  mentioned, 
has  also  determined  the  carbon-dioxide  tension  in  the  blood  of  the  pul- 

1  Bohr.  Centralbl.  f.  Physiol.,  4. 

2  Wolffberg,  Pfluger 's  Arch.,  C;  Strassburg,  ibid.;  Nussbaum,  ibid.,  7. 


CARBON-DIOXIDE   TENSION  IN   THE  BLOOD.  611 

monary  alveoli  in  a  case  simultaneously  with  the  catheterization  of  the 

hingS.       II"'    found     nearly     identical     results,     namely,     a     carbon-dioxide 
tendon  of  3.84  per  cent  and  3.81  per  cent. 

Bohr,  in  his  experiments  above  mentioned  (pane  60S),  has  arrived  at 
other  results  in  regard  to  the  carbon-dioxide  tension.  In  eleven  experi- 
ment^ with  inhalation  of  atmospheric  air  the  carbon-dioxide  tension  in  the 
arterial  blond  varied  from  0  to  3S  mm.  Hg,  and  in  five  experiments  with 
inhalation  of  air  containing  carbon  dioxide,  from  0.9  to  57.8  mm.  Hg.  A 
comparison  of  the  carbon-dioxide  tension  in  the  blood  with  the  bifurc 
air  gave  in  several  cases  a  greater  carbon-dioxide  pressure  in  the  air  of  the 
Lungs  than  in  the  blood,  and  as  maximum  this  difference  amounted  to 
17.2  mm.  in  favor  of  the  air  of  the  lungs  in  the  experiments  with  inhalation 
of  atmospheric  air.  As  the  alveolar  air  is  richer  in  carbon  dioxide  than  the 
bifurcate<  1  air,  this  experiment  unquestionably  proves,  according  to  Bohr, 
that  the  carbon  dioxide  has  migrated  against  the  high  pressure. 

In  opposition  to  these  investigations,  Fredericq,1  in  his  above-men- 
tioned experiments,  obtained  the  same  figures  for  the  carbon-dioxide  ten- 
sion in  arterial  peptone  blood  as  Pfluger  and  his  pupils  found  for  normal 
blood.  Weisgerber,2  in  Frederick's  laboratory,  has  made  experiments 
with  animals  which  respired  air  rich  in  carbon  dioxide,  and  these  experi- 
ments confirm  Pfluger 's  theory  of  respiration.  Recently  Falloise  has 
made  determinations  of  the  carbon-dioxide  tension  of  venous  blood  by  means 
of  Fredericq 's  aeorotonometer.  The  carbon-dioxide  tension  was  found 
to  equal  6  per  cent  of  an  atmosphere,  hence  somewhat  higher  than  the 
results  found  by  Pfluger \s  pupils.  The  low  figures  obtained  by  BoHB 
for  the  carbon-dioxide  tension  appear  remarkable  when  it  is  recalled  that 
Gr.vxdis  found  in  peptone  blood  which  Lahousse  and  Blachstein  3  had 
shown  was  poor  in  carbon  dioxide,  a  high  carbon-dioxide  tension. 

Graxdis  *  has  observed  that  the  tens:on  of  the  blood  gases  was  increased  by 
the  concentration  of  'he  blood.  As  the  blood  is  concentrated  by  an  increased 
giving  off  of  water  in  the  lungs,  a  temporary  rise  in  the  tension  of  the  blood  gases' 
may  take  place  here  which  may  perhaps  explain,  according  to  him,  the  great 
differences  observed  by  Bohr  and  Haloane  between  the  tension  of  the  carbon 
dioxide  of  the  blood  and  the  carbon  dioxide  in  the  air  of  the  lung  lobes. 

A  certain  importance  has  been  ascribed  to  oxygen  in  regard  to  the 
elimination  of  carbon  dioxide  in  the  lungs,  in  that  it  has  an  expelling  action 
on  the  carbon  dioxide  from  its  combinations  in  the  blood.  This  statement, 
first  made  by  Holmgren,  has  recently  found  an  advocate  in  Werigo.5 

1  See  foot-note  1,  page  609. 

2  Centralbl.  f    Physiol  .  19.  4S2. 

'Grandis.   Du  Bois-Reymond 's  Arch.,   1891;    Lahousse,  ibid.,   18S9;    Blachstein, 
ibid..  1891;    Fallrise   see  Msly's  Jabresber.,  32 
4  See  Maly's  Jahresber..  30 
6  Holmgren    Wien   Sitzungsber.,  48  Werigo,  Pfluger 's  Arch.,  51  and  52. 


612  CHEMISTRY  OF  RESPIRATION. 

This  investigator  has  made  ingenious  experiments  on  living  animals  in 
which  he  allows  both  lungs  of  the  animal  to  breathe  separately,  the  one 
with  hydrogen  and  the  other  with  pure  oxygen  or  a  gas  mixture  rich  in 
oxygen.  He  invariably  found  a  greater  carbon-dioxide  tension  in  the 
air  sucked  from  the  lungs  in  the  presence  of  oxygen,  and  he  draws  the  con- 
clusion from  his  experiments  that  the  oxygen  passing  from  the  lung  alveoli 
into  the  blood  raises  the  carbon-dioxide  tension.  According  to  Werigo, 
by  this  action  the  oxygen  is  a  powerful  factor  in  the  elimination  of  carbon 
dioxide,  and  therefore  it  is  not  necessary  to  assume  a  specific  action  of  the 
lung  itself  in  these  processes. 

Zuntz  *  has  suggested  important  objections  to  the  conclusions  of 
Werigo,  but  they  have  not  been  substantiated  by  experiment;  hence 
the  question  is  still  open. 

The  picture  in  regard  to  the  carbon-dioxide  elimination  in  the  lungs 
is  not  yet  clear,  and  we  must  wait  for  further  light  upon  it. 

From  what  has  been  said  above  (page  604)  in  regard  to  the  internal 
respiration  one  can  conclude  that  it  consists  chiefly  in  that  in  the  capil- 
laries the  oxygen  passes  from  the  blood  into  the  tissues,  while  the  carbon 
dioxide  passes  from  the  tissues  into  the  blood. 

The  assertion  of  Estor  and  Saint  Pierre  that  the  quantity  of  oxygen 
in  the  blood  of  the  arteries  decreases  with  the  remoteness  from  the  heart 
has  been  shown  to  be  incorrect  by  Pfluger,2  and  the  oxygen  tension  in  the 
blood  on  entering  the  capillaries  must  be  higher.  As  compared  with  the 
capillaries  the  tissues  are  to  be  considered  as  nearly  or  entirely  free  from 
oxygen,  and  in  regard  to  the  oxygen  a  considerable  difference  in  pressure 
must  exist  between  the  blood  and  tissues.  The  possibility  that  this  differ- 
ence in  pressure  is  sufficient  to  supply  the  tissues  with  the  necessary  quantity 
of  oxygen  is  hardly  to  be  doubted. 

In  regard  to  the  carbon-dioxide  tension  in  the  tissue  it  must  be  assumed 
h  priori  that  it  is  higher  than  in  the  blood.  This  is  found  to  be  true. 
Strassburg  3  found  in  the  urine  of  dogs  and  in  the  bile  a  carbon-dioxide 
tension  of  9  per  cent  and  7  per  cent  of  an  atmosphere,  respectively.  The 
same  experimenter  has,  further,  injected  atmospheric  air  into  a  ligatured 
portion  of  the  intestine  of  a  living  dog  and  analyzed  the  air  taken  out  after 
some  time.  He  found  a  carbon-dioxide  tension  of  7.7  per  cent  of  an  atmos- 
sphere.  The  carbon-dioxide  tension  in  the  tissues  is  considerably  greater 
than  in  the  venous  blood,  and  there  is  no  opposition  to  the  view  that  the 
carbon  dioxide  simply  diffuses  from  the  tissues  into  the  blood  according  to 
the  laws  of  diffusion. 


1  Pfliiger's  Arch.,  52 

2  Estor  and  Saint  Pierre  with  Pfluger  in  Pfliiger's  Arch.,  1. 
8  Pfluger 's  Arch.,  6. 


METHODS  OP  EXPERIMENTATION.  G13 

That  a  true  secretion  of  gases  occurs  in  animals  follows  from  the  composition 
and  behavior  of  the  Eases  in  the  Bwimming-bladder  of  fishes.  These  gases  con- 
of  oxygen  and  nitrogen  with  only  small  quantities  of  carbon  dioxide.  In 
fishes  which  do  not  live  at  any  great  depth  the  quantity  of  oxygen  is  ordinarily 
as  high  as  in  the  atmosphere,  while  fishes  which  live  at  great  depths  may,  accord- 
ing to  BlOT  and  others,  contain  considerably  more  oxygen  and  even  above  SO  per 
cent.  MoREATJ  has  also  found  that  after  emptying  the  swimming-bladder  by 
means  of  a  trocar  new  air  collected  after  a  time,  and  this  air  was  richer  in  oxygen 
than  the  atmospheric  air  and  contained  even  85  per  cent  oxygen.  BoHB,  who 
has  proved  and  confirmed  these  statements,  also  found  that  this  collection  is 
under  the  influence  of  the  nervous  system,  because  on  the  section  of  certain 
branches  of  the  pneumogastrie  nerve  it  is  discontinued.  It  is  beyond  dispute  that 
there  is  here  a  secretion  and  not  a  diffusion  of  oxygen.  Recently  Jaegeb  '  has 
given  a  further  explanation  as  to  the  secretory  activity  of  the  swimming-bladder. 

Several  methods  have  been  suggested  for  the  study  of  the  quantitative 
relationship  of  the  raspiratory  exchange  of  gas.  The  reader  must  be  referred 
to  other  text-books  for  more  details  as  to  these  methods,  and  we  will  here 
only  mention  the  chief  features  of  the  most  important  method.-. 

Regnault  and  Reiset's  Method.  According  to  this  method  the  animal  or 
person  experimented  upon  is  allowed  to  respire  in  an  enclosed  space.  The 
carbon  dioxide  is  removed  from  the  air,  as  it  forms,  by  strong  caustic  alkali,  from 
which  the  quantity  may  be  determined,  while  the  oxygen  is  replaced  continually 
by  exactly  measured  quantities.  This  method,  which  also  makes  possible  a  direct 
determination  of  the  oxygen  used  as  well  as  the  carbon  dioxide  produced,  has  since 
been  modified  by  other  investigators,  such  as  Pfliger  and  his  pupils  Beegen 
and  Nowak,  and  Hoppe-Seyler,  Rosenthal,  and  Zuntz.2 

Pettexkofkr's  Method.  According  to  this  method  the  individual  to  be 
experimented  upon  breathes  in  a  room  through  which  a  current  of  atmospheric 
air  is  passed.  The  quantity  of  air  passed  through  is  carefully  measured.  As  it 
is  impossible  to  analyze  all  the  air  made  to  pass  through  the  chamber,  a  small 
fraction  of  this  air  is  diverted  into  a  branch  line  during  the  entire  experiment, 
carefully  measured,  and  the  quantity  of  carbon  dioxide  and  water  determined. 
From  the  composition  of  this  air  the  quantity  of  water  and  carbon  dioxide  con- 
tained in  the  large  quantity  of  air  made  to  pa<s  through  the  chamber  can  be 
calculated.  The  consumption  of  oxygen  cannot  be  directly  determined  in  this 
method,  but  may  be  calculated  indirectly  by  difference,  which  is  a  defect  in  this 
method.  The  large  respiration  apparatus  of  SoNDEN  and  TlGEBSTEDT  as  well  as  of 
Atw.vter  and  Ros.v  3  are  based  upon  this  principle. 

Speck's  Method.4  For  briefer  experiments  on  man  Spkck  has  u>ed  the 
following:  He  breathes  into  two  spirometer-receivcrs,  on  which  the  gis-volume 
can  he  read  off  very  accurately,  through  a  mouthpiece  with  two  valves,  closing  the 
nose  with  a  clamp.  The  air  from  one  of  the  spirometers  is  inhaled  through  one 
valve  and  the  expired  air  passes  through  the  other  into  the  other  spirometer. 

1  Biot,  see  Hermann's  Handhuch  d.  Physiol.,  4,  Thl.  2,  151:  Moreau,  Compt. 
rend.,  57;  Bohr,  Journ.  of  Physiol.,  15.  See  also  Hiifner,  Du  Bois-Revmond'-  Arch., 
1892;   Jaeger,  Pfluger's  Arch.,  94. 

*See  Zuntz  in  Hermann's  Handhuch,  4,  Thl.  2,  and  Hoppe-Seyler,  Zeitschr.  f. 
physiol.  Chem.,  19;  Rosenthal,  Arch.  f.  (Anat.  u.)  Physiol.,  1902;  Zuntz,  Verhandl. 
d.  Berl.  physiol.  Gesellsch.,  1901. 

'J  IVttenkofer^s  method;  see  Zuntz,  1.  c. ;  Sonden  and  Tiperstedt,  Skand.  Arch.  f. 
Physiol.,  6;   Atwater  and  Rosa,  Bull,  of  Dept.  of  Agriculture.  63.     Washington. 

*  Speck,  Physiologie  des  menschlichen  Athmens.     Leipzig,  1S92. 


614  CHEMISTRY  OF  RESPIRATION. 

By  means  of  a  rubber  tube  connected  with  the  expiration-tube  an  accurately 
measured  part  of  the  expired  air  may  be  passed  into  an  absorption-tube  and 
analyzed. 

Zuntz  and  Geppert's  Method.1  This  method,  which  has  been  improved 
by  Zuntz  and  his  pupils  from  time  to  time,  consists  in  the  following :  The  individual 
being  experimented  upon  inspires  pure  atmospheric  air  through  a  very  wide 
feed-pipe  leading  from  the  open  air,  the  inspired  and  the  expired  air  being  separated 
by  two  valves  (human  f  ubjects  breathe  with  closed  nose  by  means  of  a  soft-rubber 
mouthpiece,  animals  through  an  air-t'ght  tracheal  canula).  The  volume  of  the 
expired  air  is  measured  by  a  gas-meter  and  an  aliquot  part  of  this  air  collected 
and  the  quantity  of  carbon  dioxide  and  oxygen  determined.  As  the  composition 
o  the  atmospheric  air  can  be  considered  as  constant  within  a  certain  limit,  the 
production  of  carbon  dioxide  as  well  as  the  consumption  of  oxygen  may  be  readily 
calculated  (see  the  works  of  Zuntz  and  his  pupils). 

Hanriot  and  Richet's  method2  is  characterized  by  its  simplicity.  These 
investigators  allow  the  total  air  to  pass  through  three  gasometers,  one  after  the 
other.  The  first  measures  the  inspired  air,  whose  composition  is  known.  The 
second  gasometer  measures  the  expired  air,  and  the  third  the  quantity  of  the 
expired  air  after  the  carbon  d' oxide  has  been  removed  by  a  suitable  apparatus. 
The  quantity  of  carbon  dioxide  produced  and  the  oxygen  consumed  can  be  readily 
calculated  from  these  data. 

Appendix. 
The  Lungs  and  their  Expectorations. 
Besides  proteid  bodies  and  the  albuminoids  of  the  connective-substance 
group,  lecithin,  taurin  (especially  in  ox-lungs),  uric  acid,  and  inosite  have 
been  found  in  the  lungs.  Poulet  3  claims  to  have  found  a  special  acid, 
which  he  has  called  pulmotartaric  acid,  in  the  lung-tissue.  Glycogen 
occurs  abundantly  in  the  embryonic  lung,  but  is  absent  in  the  adult  organ. 
The  proteolytic  enzymes  also  belong  to  the  physiological  constituents  of 
the  lungs.  They  are  active  in  the  autolysis  of  the  lungs  ( Jacob y)  as  well 
as  in  the  solution  of  pneumonic  infiltrations  (Fr.  Muller  4). 

The  black  or  dark-brown  pigment  in  the  lungs  of  human  beings  and  domestic 
animals  consists  chiefly  of  carbon,  which  originates  from  the  soot  in  the  air.  The 
pigment  may  in  part  also  consist  of  melanin.  Besides  carbon,  other  bodies,  such 
as  iron  oxide,  silicic  acid,  and  clay,  may  be  deposited  in  the  lungs,  being  inhaled 
as  dust. 

Among  the  bodies  found  in  the  lungs  under  pathological  conditions  must 
be  specially  mentioned  albumoses  (and  peptones?)  in  pneumonia  and  sup- 
puration, glycogen,  a  slightly  dextrorotatory  carbohydrate  differing  from 
glycogen  found  by  Pouchet  in  consumptives,  and  finally  also  cellulose, 
which,  according  to  Freuxd,5  occurs  in  the  lungs,  blood,  and  pus  of  persons 
with  tuberculosis. 

1  Pfluger's  Arch.,  42.  See  also  Magnus-Levy  in  Pfliiger's  Arch.,  55,  10,  in  which 
the  work  of  Zuntz  and  his  pupils  is  cited. 

2  Compt.  rend.,  104. 

3  Cited  from  Maly's  Jahresber.,  18,  248. 

4Jacoby,  Zeitschr.  f.  physiol.  Chem.,  33;  Muller,  Verhandl.  d.  Kongress.  f.  inn. 
Medizin,  1902. 

5  Pouchet,  Compt.  rend.,  96;    Freund,  cited  from  Maly's  Jahresber,  16,  471. 


LUNGS  AND   Til  KIR  EXPECTORATIONS.  615 

C.  W.  Schmidt  found  in  1000  grams  of  mineral  bodies  from  the  normal 
human  lung  the  following:  XaCl  130,  KjO  13,  Xa,<  >  L95,  CaO  19,  MgO  19, 
Fe203  32,  P206  485,  S03  8,  and  sand  L34  grams.  According  to  Oidtmaxx  ' 
the  lungs  of  a  1-t-day-old  child  contained  790.05  p.  m.  water,  198.19  p.  m. 
organic  bodies,  and  5. 70  p.  m.  inorganic  bodies. 

The  sputum  is  a  mixture  of  the  mucous  secretion  of  the  respiratory 
passages,  of  saliva  and  buccal  mucus.  Because  of  this  its  composition  is 
very  variable,  especially  under  pathological  conditions  when  various  prod- 
ucts mix  with  it.  The  chemical  constituents  are,  besides  the  mineral 
substances,  chiefly  mucin  with  a  little  proteid  and  nuclein  substance. 
Under  pathological  conditions  albumoses  and  peptone  (?),  which  are  prob- 
ably produced  by  bacterial  action  or  by  autolysis  (Wanner,  Simon  -), 
volatile  fatty  acids,  glycogen,  Charcot's  crystals,  and  also  crystals  of 
cholesterin,  haematoidin,  tyrosin,  fat  and  fatty  acids,  triple  phosphates, 
etc.,  have  bei^n  found. 

The  form  constituents  are,  under  physiological  circumstances,  epithe- 
lium-cells of  various  kinds,  leucocytes,  sometimes  also  red  blood-corpuscles 
and  various  kinds  of  fungi.  In  pathological  conditions  elastic  fibres, 
spiral  formations  consisting  of  a  mucin-like  substance,  fibrin  coagulum, 
pus,  pathogenic  microbes  of  various  kinds,  and  the  above-mentioned 
crystals  occur. 

'Schmidt,  cited  from  v.  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  727;  Oidtmann, 
ibid.,  732. 

1  Wanner,  Deutsch.  Arch.  f.  klin.  Med.,  75;  Simon,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 


CHAPTER  XVIII. 

METABOLISM  WITH  VARIOUS  FOODS,  AND  THEIR  NECESSITY 

TO  MAN. 

The  conversion  of  chemical  energy  into  heat  and  mechanical  work,, 
which  characterizes  animal  life,  leads,  as  previously  stated  in  Chapter  I, 
to  the  formation  of  relatively  simple  compounds — carbon  dioxide,  urea,, 
etc. — which  leave  the  organism,  and  which,  moreover,  being  very  poor  in 
energy,  are  for  this  reason  of  little  or  no  value  for  the  body.  It  is 
therefore  absolutely  necessary  for  the  continuance  of  life  and  the  normal 
course  of  the  functions  of  the  body  that  the  organism  and  its  different 
tissues  should  be  supplied  with  new  material  to  replace  that  which  has 
been  exhausted.  This  is  accomplished  by  means  of  food.  Those  bodies 
are  designated  as  food  which  have  no  injurious  action  upon  the  organism 
and  which  serve  as  a  source  of  energy  and  can  replace  those  constituents 
of  the  body  that  have  been  consumed  in  metabolism  or  that  can  prevent 
or  diminish  the  consumption  of  such  constituents. 

Among  the  numerous  dissimilar  substances  which  man  and  animals 
take  with  the  food  all  cannot  be  equally  necessary  or  have  the  same  value. 
Some  perhaps  are  unnecessary,  while  others  may  be  indispensable.  We 
have  learned  by  direct  observation  and  a  wide  experience  that  besides  the 
oxygen,  which  is  necessary  for  oxidation,  the  essential  foods  for  animals  in 
general,  and  for  man  especially,  are  water,  mineral  bodies,  proteins,  carbo- 
hydrates, and  fats. 

It  is  also  apparent  that  the  various  groups  of  foodstuffs  necessary  for 
the  tissues  and  organs  must  be  of  varying  importance;  thus,  for  instance, 
water  and  the  mineral  bodies  have  another  value  than  the  organic  foods, 
and  these  again  must  differ  in  importance  among  themselves.  The  knowl- 
edge of  the  action  of  various  nutritive  bodies  on  the  exchange  of  material 
from  a  qualitative  as  well  as  a  quantitative  point  of  view  must  be  of  funda- 
mental importance  in  determining  the  value  of  different  nutritive  sub- 
stances relative  to  the  demands  of  the  body  for  food  under  various  condi- 
tions, and  also  in  deciding  many  other  questions — for  instance,  the  proper 
mitrition  for  an  individual  in  health  and  in  disease. 

Such  knowledge  can  only  be  attained  by  a  series  of  systematic  and 
thorough  observations,  in  which  the  quantity  of  nutritive  material,  relative 
to  the  weight  of  the  body,  taken  and  absorbed  in  a  given  time  is  compared 

616 


EXCRETA  OF   THE  ORGANISM.  617 

With  the  quantity  of  final  metabolic  products  which  leave  the  organism  at 
the  sitine  time.  Researches  of  this  kind  have  been  made  by  several  investi- 
gators, but  above  all  should  be  mentioned  those  made  by  BlSCHOPP  and 
Voit,  by  Pettenkofer  and  Voit,  and  by  Voit  and  his  pupils,  and  by 

RUBNER. 

It  i<  absolutely  necessary  in  researches  on  the  exchange  of  materia]  to 
be  able  to  collect,  analyze,  and  quantitatively  estimate  the  excreta  of  the 
organism,  so  that  they  may  be  compared  with  the  quantity  and  composition 
of  the  nutritive  bodies  ingested.  In  the  first  place,  one  must  know  what 
the  habitual  excreta  of  the  body  are  and  in  what  way  these  bodies  leave  the 
organism.  One  must  also  have  trustworthy  methods  for  the  quantitative 
estimation  of  the  same. 

The  organism  may,  under  physiological  conditions,  be  exposed  to  acci- 
dental or  periodic  losses  of  valuable  material — such  losses  as  only  occur  in 
certain  individuals,  or  in  the  same  individual  only  at  a  certain  period;  for 
instance,  the  secretion  of  milk,  the  production  of  eggs,  the  ejection  of 
semen  or  menstrual  blood.  It  is  therefore  apparent  that  these  losses  can 
be  the  subject  of  investigation  and  estimation  only  in  special  cases. 

The  regular  and  constant  excreta  of  the  organism  are  of  the  very  great- 
est importance  in  the  study  of  metabolism.  To  these  belong,  in  the  first 
place,  the  true  final  metabolic  products — carbon  dioxide,  urea  (uric  acid, 
hippuric  acid,  creatinine,  and  other  urinary  constituents),  and  a  part  of  the 
water.  The  remainder  of  the  water,  the  mineral  bodies,  and  those  secre- 
tions or  tissue  constituents — mucus,  digestive  fluids,  sebum,  perspiration, 
and  epidermal  formations — which  are  either  poured  into  the  intestinal 
tract,  or  secreted  from  the  surface  of  the  body,  or  broken  off  and  thereby 
lost  to  the  body,  also  belong  to  the  constant  excreta. 

The  remains  of  food,  sometimes  indigestible,  sometimes  digestible  but  not  acted 
upon,  which  are  contained  in  the  faeces,  and  which  vary  considerably  in  quantity 
and  composition  with  the  nature  of  the  food,  also  belong  to  the  excreta  of  the 
organism.  Even  though  these  remains,  which  are  never  absorbed  and  therefore  are 
never  constituents  of  the  animal  fluids  or  tissues,  cannot  be  considered  as  excreta 
of  the  body  in  a  strict  sense,  still  their  quantitative  estimation  is  absolutely  neces- 
sary in  certain  experiments  on  the  exchange  of  material. 

The  determination  of  the  constant  loss  is  in  some  cases  accompanied  with 
the  greatest  difficulties.  The  loss  from  the  detached  epidermis,  from  the  secret  inn 
of  the  sebaceous  glands,  etc.,  cannot  be  determined  with  exactness  without  diffi- 
culty, and  therefore — as  they  do  not  occasion  any  appreciable  loss  because  of 
their  small  quantity — they  need  not  be  considered  in  quantitative  experiments 
on  metabolism.  This  also  applies  to  the  constituents  of  the  mucus,  bile,  pan- 
creatic and  intestinal  juices,  etc.,  occurring  in  the  contents  of  the  intestine,  and 
which,  leaving  the  body  with  the  fseces,  cannot  be  separated  from  the  other  eon- 
tents  of  the  intestine  and  therefore  cannot  be  quantitatively  determined  sepa- 
rately. The  uncertainty  which,  because  of  the  intimated  difficulties,  attaches 
itself  to  the  results  of  the  experiments  is  very  small  as  compared  to  the  variation 
which  is  caused  by  different  individualities,  different  modes  of  living,  different 
food-,  etc.  No  genera]  but  only  approximate  values  can  therefore  be  g'ven  for 
the  constant  excreta  of  the  human  body. 


61S  METABOLISM  WITH   VARIOUS  FOODS 

The  following  figures  represent  the  quantity  of  excreta  for  twenty-four 
hours  from  a  grown  man,  weighing  60-70  kilos,  on  a  mixed  diet.  The 
numbers  are  compiled  from  the  results  of  different  investigators. 

Grams. 

Water 2500-3500 

Salts  (with"  the  urine) 20-30 

Carbon  dioxide 750-900 

Urea.  . 20-40 

Other  nitrogenous  urinary  constituents 2-5 

Solids  in  the  excrements 20-50 

These  total  excreta  are  approximately  divided  among  the  various 
excretions  in  the  following  way;  but  still  it  must  not  be  forgotten  that 
this  division  may  vary  to  a  great  extent  under  various  external  circum- 
stances: by  respiration  about  32  per  cent,  by  the  evaporation  from  the 
skin  17  per  cent,  with  the  urine  46-47  per  cent,  and  with  the  excrements 
5-9  per  cent.  The  elimination  by  the  skin  and  lungs,  which  is  sometimes 
differentiated  by  the  name  " per spir alio  insensibilis"  from  the  visible 
elimination  by  the  kidneys  and  intestine,  is  on  an  average  about  50  per 
cent  of  the  total  elimination.  This  proportion,  quoted  only  relatively, 
is  subject  to  considerable  variation,  because  of  the  great  difference  in 
the  loss  of  water  through  the  skin  and  kidneys  under  different  circum- 
stances. 

The  nitrogenous  constituents  of  the  excretions  consist  chiefly  of  urea, 
or  uric  acid  in  certain  animals,  and  the  other  nitrogenous  urinary  con- 
stituents. A  disproportionately  large  part  of  the  nitrogen  leaves  the  body 
with  the  urine,  and,  as  the  nitrogeneous  constituents  of  this  excretion  are 
final  products  of  the  metabolism  of  proteids  in  the  organism,  the  quantity 
of  proteids  catabolized  in  the  body  may  be  easily  calculated  by  multiplying 
the  quantity  of  nitrogen  in  the  urine  by  the  coefficient  6.25  (1T°ir  —  6.25),  if  it  is 
admitted  that  the  proteids  contain  in  round  numbers  16  per  cent  of  nitrogen. 

Still  another  question  is  whether  the  nitrogen  leaves  the  body  only  with 
the  urine  or  by  other  channels.  The  latter  is  habitually  the  case.  The  dis- 
charges from  the  intestine  always  contain  some  nitrogen,  which  as  stated 
in  Chapter  IX  consists  in  part  of  non-absorbed  remnants  of  the  food,  but 
in  chief  part  and  sometimes  entirely  of  constituents  of  the  epithelium  and 
the  secretions.  Under  these  circumstances  it  is  apparent  that  one  cannot 
give  any  exact  figures  which  are  valid  for  all  cases  for  that  part  of  the  nitro- 
gen of  the  excrements  which  originates  from  the  digestive  tract  and  from 
the  digestive  fluids.  It  may  not  only  vary  in  different  individuals,  but 
also  in  the  same  individual  after  more  or  less  active  secretion  and  absorp- 
tion. In  the  attempts  made  to  determine  this  part  of  the  nitrogen  of 
the  excrements  it  has  been  found  that  in  man,  on  non-nitrogenous  or  nearly 
nitrogen-free  food,  it  amounts  in  round  numbers  to  somewhat  less  than 
1  gram  per  twenty-four  hours  (Rieder,  Rubner).     Even  with  such  food 


NITROGENOUS  EQUILIBRIUM.  019 

the  absolute  quantity  of  nitrogen  eliminated  by  the  faeces  increases  with 

the  quantity  of  food  because  of  the  accelerated  digestion  (TsUBOI  '),  and  is 
greater  than  In  starvation.  Mi'llek2  found  in  his  observations  on  the  faster 
Cetti  that  only  0.2  grain  nitrogen  was  derived  from  the  intestinal  canal. 

The  quantity  of  nitrogen  which  leaves  the  body  under  normal  circum- 
stances by  means  of  the  hair  and  nails,  with  the  scaling  off  of  the  skin,  and 
with  the  perspiration  cannot  be  accurately  determined.  It  is  neverthe- 
less so  small  thai  it  may  be  ignored.  Only  in  profuse  sweating  need  the 
elimination  by  this  channel  be  taken  into  consideration. 

The  view  was  formerly  held  that  in  man  and  carnivora  an  elimination 
of  gaseous  nitrogen  took  place  through  the  skin  and  lungs,  and  because  of 
this,  on  comparing  the  nitrogen  of  the  food  with  that  of  the  urine  and 
faeces,  a  nitrogen  deficit  occurred  in  the  visible  elimination. 

This  question  has  been  the  subject  of  much  discussion  and  of  numerous 
investigations.3  These  investigations  have  shown  that  the  above  assump- 
tion is  unfounded,  and  moreover  several  investigators,  especially  Petten- 
kofer  and  Voit,  and  Gruber,4  have  shown  by  experiments  on  man  and 
animals  that  with  the  proper  quantity  and  quality  of  food  the  body  can 
be  brought  into  nitrogenous  equilibrium,  in  which  the  quantity  of  nitrogen 
voided  with  the  urine  and  faeces  is  equal  or  nearly  equal  to  the  quantity 
contained  in  the  food.  Undoubtedly  we  must  admit  with  Vorr  thai  a 
deficit  of  nitrogen  does  not  exist,  or  it  is  so  insignificant  that  in  experi- 
ments upon  metabolism  it  need  not  be  considered.  Ordinarily,  in  investi- 
gations on  the  catabolism  of  proteids  in  the  body,  it  is  only  necessary  to 
consider  the  nitrogen  of  the  urine  and  faeces,  but  it  must  be  remarked  that 
the  nitrogen  of  the  urine  is  a  measure  of  the  extent  of  the  catabolism  of 
the  proteids  in  the  body,  while  the  nitrogen  of  the  faeces  (after  deducting 
about  1  gram  on  a  mixed  diet)  is  a  measure  of  the  non-absorbed  part  of  the 
nitrogen  of  the  food.  The  nitrogen  of  the  food,  as  well  as  of  the  excreta. 
is  generally  determined  by  Kjeldahl's  method. 

In  the  oxidation  of  the  proteids  in  the  organism  their  sulphur  is  oxidized 
into  sulphuric  acid,  and  on  this  depends  the  fact  that  the  elimination  of 
sulphuric  acid  by  the  urine,  which  in  man  is  only  to  a  small  extent  derived 
from  the  sulphates  of  the  food,  makes  nearly  equal  variations  with  the  elimi- 
nation of  nitrogen  by  the  urine.  If  the  amount  of  nitrogen  and  sulphur 
in  the  proteids  is  considerd  as  16  per  cent  and  1  per  cent  respectively, 
then  the  proportion  between  the  nitrogen  of  the  proteids  and  the  sulphuric 

1  Rieder,  Zeitschr.  f.  Biologie,  20;   Rubner,  ibid.,  IS;  Tsuboi,  ibid.,  35. 

1  Berlin,  klin.  Wochenschr. ,  1S87. 

3  See  Regnault  and  Reiset,  Annal.  d.  ehim.  et  phys.  (3),  26,  and  Annal.  d.  Chem. 
u.  Pharm.,  73;  Seegen  and  Nowak,  Wien.  Sitzungsber.,  71,  and  Pfliiger's  Arch.,  2.">; 
Pettenkofer  and  Voit,  Zeistchr.  f.  Biologie,  10;    Leo,  Pfliiger's  Arch.,  2(5. 

'Pettenkofer  and  Voit,  in  Hermann's  Handbuch,  6,  Thl.  1;  Gruber,  Zeitschr.  f. 
Biologie,  16  and  19. 


620  METABOLISM  WITH  VARIOUS  FOODS. 

acid,  H2S04,  produced  by  their  combustion  is  in  the  ratio  5.2:1,  or  about 
the  same  as  in  the  urine  (see  page  532) .  The  determination  of  the  quantity 
of  sulphuric  acid  eliminated  in  the  urine  gives  us  an  important  means 
of  controlling  the  extent  of  the  transformation  of  proteids,  and  such  a 
control  is  especially  important  in  cases  in  which  it  is  expected  to  study 
the  action  of  certain  nitrogenous  non-albuminous  bodies  on  the  metabolism 
of  proteids.  A  determination  of  the  nitrogen  alone  is  not  sufficient  in 
such  cases.  A  perfectly  positive  measure  of  the  proteid  catabolism  can- 
not be  made  from  the  sulphuric  acid  of  the  urine,  as  the  various  protein 
substances  have  a  rather  variable  sulphur  content,  and  on  the  other  hand 
also  a  variable  quantity  of  the  sulphur  in  the  urine  exists  as  so-called 
neutral  sulphur. 

The  pseudonucleins,  as  well  as  the  true  nucleins,  may  be  absorbed  more 
or  less  completely  from  the  intestinal  tract  and  then  assimilated  (Gum- 
xi ch,  Sandmeyer,  Marcuse,  Rohmann,  and  Steinitz,  Loewi,1  and 
others).  On  the  other  hand,  the  phosphorized  protein  substances,  leci- 
thins and  protagons,  are  also  decomposed  within  the  body,  and  their  phos- 
phorus s  chiefly  eliminated  as  phosphoric  acid  and  also  in  part  as  organic 
phosphorus  (see  page  525).  For  these  reasons  the  phosphorus  is  of 
great  importance  in  certain  investigations  on  metabolism. 

If  it  is  found,  on  comparing  the  nitrogen  of  the  food  with  that  of  the 
urine  and  faeces,  that  there  is  an  excess  of  the  first,  this  means  that  the 
body  has  increased  its  stock  of  nitrogenous  substances — proteids.  If,  on 
the  contrary,  the  urine  and  faeces  contain  more  nitrogen  than  the  food  taken 
at  the  same  time,  this  denotes  that  the  body  is  giving  up  part  of  its  nitro- 
gen— that  is,  a  part  of  its  own  proteids  has  been  decomposed.  We  can, 
from  the  quantity  of  nitrogen,  as  above  stated,  calculate  the  corresponding 
quantity  of  proteids  by  multiplying  by  6.25.  Usually,  according  to  Voit's 
proposition,  the  nitrogen  of  the  urine  is  not  calculated  as  decomposed 
proteids,  but  as  decomposed  muscle-substance  or  flesh.  Lean  meat  con- 
tains on  an  average  about  3.4  per  cent  nitrogen;  hence  each  gram  of  nitro- 
gen of  the  urine  corresponds  in  round  numbers  to  about  30  grams  of  flesh. 
The  assumption  that  lean  meat  contains  3.4  per  cent  nitrogen  is  arbitrary, 
and  the  relationship  of  N:C  in  the  proteids  of  dried  meat,  which  is  of 
great  importance  in  certain  experiments  on  metabolism,  is  given  differently 
by  various  experimenters,  namely,  1:3.22 — 1:3.68.  Argutinsky  found  in 
beef,  after  complete  removal  of  fat  and  subtraction  of  glycogen,  that  the 
relationship  was  1:3.24  (sec  Chapter  XI). 

1  In  regard  to  the  investigations  on  the  metabolism  of  phosphorus  and  the  methods 
used  therein,  see  Steinitz,  Pfluger's  Arch.,  72;  Zadik,  ibid.,  77;  Leipziger,  ibid.,  78; 
Oertel  Zeitschr.  f.  physiol.  Chem.,  2G;  Mandel  and  Oertel,  Bull.  Med.  Sciences,  N.  Y. 
Univ.,  l,and  Ehrlich,  Inaug.-Diss.,  Breslau,  1900;  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm., 
4.V  On  the  absorption  of  casein,  see  Poda,  Prausnitz,  Micko,  and  P.  Miiller,  Zeitschr.  f. 
Biologie,  39. 


CALCULATION  OF  THE  EXTENT  OF  CATABOLISM.  621 

The  carbon  loaves  the  body  chiefly  as  carbon  dioxide,  which  is  elimi- 
nated by  the  lungs  and  skin.  The  remainder  of  the  carbon  is  excreted  in 
the  urine  and  faeces  in  the  form  of  organic  compounds,  in  which  the  quan- 
tity of  carbon  <;an  be  determined  by  elementary  analysis.  It  used  to  be 
considered  sufficient  to  calculate  the  quantity  of  carbon  in  the  urine  from 
the  quantity  of  nitrogen  according  to  the  relationship  N:C=  1:0.67.  This 
does  not  seem  to  be  trustworthy,  as  this  relationship  varies  and  depends, 
according  to  Tangl  and  Pfluger,1  upon  the  kind  of  food.  Taxgl  has 
shown  that  the  richer  the  food  is  in  carbohydrates  the  more  carbon  and 
heat  of  combustion  per  gram  of  nitrogen  does  the  urine  contain.  He 
found  the  following  for  1  gram  of  nitrogen  in  the  urine:  With  diet  rich 
in  fat  0.747  gram  C  and  9.22  Calories;  for  carbohydrate-rich  diet  he  found 
0.963  gram  C  and  11.67  Calories. 

The  quantity  of  gaseous  carbon  dioxide  eliminated  may  be  determined 
by  means  of  Pettenkofer's  respiration  apparatus  or  by  other  methods. 
By  multiplying  the  quantity  of  carbon  dioxide  found  by  0.273  one  obtains 
the  quantity  of  carbon  eliminated  as  C02.  If  the  total  quantity  of  carbon 
eliminated  in  various  ways  is  compared  with  the  carbon  contained  in  the 
food  some  idea  can  be  obtained  as  to  the  transformation  of  the  carbon 
compounds.  If  the  quantity  of  carbon  in  the  food  is  greater  than  in  the 
excreta,  then  the  excess  is  deposited;  while  if  the  reverse  be  the  case  it 
shows  a  corresponding  loss  of  body  substance. 

The  nature  of  the  substances  here  deposited  or  lost,  whether  they  con- 
sist of  proteids,  fats,  or  carbohydrates,  is  learned  from  the  total  quantity  of 
the  nitrogen  of  the  excretions.  The  corresponding  quantity  of  proteids  may 
be  calculated  from  the  quant  ty  of  nitrogen,  and,  as  the  average  quantity 
of  carbon  in  the  proteids  is  known,  the  quantity  of  carbon  which  corre- 
sponds to  the  decomposed  proteids  may  be  easily  ascertained.  If  the 
quantity  of  carbon  thus  found  is  smaller  than  the  quantity  of  the  total 
carbon  in  the  excreta,  it  is  then  obvious  that  some  other  nitrogen-free  sub- 
stance has  been  consumed  besides  the  proteids.  If  the  quantity  of  carbon 
in  the  proteids  is  considered  in  round  numbers  as  53  per  cent,2  then  the 
relation  between  carbon  (53)  and  nitrogen  (16)  is  as  3.3:1.  If  the  total 
quantity  of  nitrogen  eliminated  is  multiplied  by  3.3,  the  excess  of  carbon  in 
the  eliminations  over  the  product  found  represents  the  carbon  of  the  decom- 
posed non-nitrogenous  compounds.  For  instance,  in  the  case  of  a  person 
experimented  upon,  10  grams  of  nitrogen  and  200  grams  of  carbon  were  elimi- 
nated in  the  course  of  24  hours;  then  these  62.5  grams  of  proteid  correspond 
to  33  grams  of  carbon,  and  the  difference,  200  — (3.3x10)  =  167.  represents 
the  quantity  of  carbon  in  the  decomposed  non-nitrogenous  compounds.  If 
we  start  from  the  simplest  case,  starvation,  where  the  body  lives  at  the 


1  Pfluger,  Pfliiger's  Arch.,  "9;  Tangl,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Supplbd. 

2  This  figure  is  perhaps  a  little  too  high. 


622  METABOLISM  WITH   VARIOUS  FOODS. 

expense  of  its  own  substance,  then,  since  the  quantity  of  carbohydrates  as 
compared  with  the  fats  of  the  body  is  extremely  small,  in  such  cases  in 
order  to  avoid  mistakes  the  assumption  must  be  made  that  the  person 
experimented  upon  has  used  only  fat  and  proteids.  As  animal  fat  con- 
tains on  an  average  76.5  per  cent  carbon,  the  quantity  of  transformed  fat 

may  be  calculated  by  multiplying  the  carbon  by  — —  =  1.3.     In  the  case  of 

the  above  example,  the  person  experimented  upon  would  have  used  62.5 
grams  of  proteids  and  167X1.3  =  217  grams  of  fat  of  his  own  body  in  the 
course  of  the  twenty-four  hours. 

Starting  from  the  nitrogen  balance,  it  can  be  calculated  in  the  same  way 
whether  an  excess  of  carbon  in  the  food  as  compared  with  the  quantity  of 
carbon  in  the  excreta  is  retained  by  the  body  as  proteids  or  fat  or  as  both. 
On  the  other  hand,  with  an  excess  of  carbon  in  the  excreta  one  can  deter- 
mine how  much  of  the  loss  of  the  substance  of  the  body  is  due  to  a  con- 
sumption of  the  proteids  or  of  fat  or  of  both. 

The  quantity  of  water  and  mineral  bodies  voided  with  the  urine  and 
faeces  can  easily  be  determined.  The  quantity  of  water  eliminated  by  the 
skin  and  lungs  may  be  directly  estimated  by  means  of  Pettenkofer's 
apparatus.  The  quantity  of  oxygen  taken  up  is  calculated  as  the  differ- 
ence between  the  weight  of  the  individual  before  the  experiment  plus  all 
the  directly  determined  substances  ingested,  and  the  final  weight  of  the 
individual  plus  all  his  excreta. 

The  oxygen  may  also  be  determined  directly,  according  to  Regnault- 
Rieset's  method,  or  in  other  ways,  and  such  a  determination  with  the 
simultaneous  estimation  of  the  carbon  dioxide  eliminated  is  of  great  impor- 
tance in  the  study  of  metabolism.1 

On  comparing  the  inspired  and  the  expired  air  we  learn,  on  measuring 
them  when  dry  and  at  the  same  temperature  and  pressure,  that  the  volume 
of  the  expired  air  is  less  than  that  of  the  inspired  air.  This  depends  upon 
the  fact  that  not  all  of  the  oxygen  appears  again  in  the  expired  air  as  car- 
bon dioxide,  because  it  is  not  only  used  in  the  oxidation  of  carbon,  but 
also  in  part  in  the  formation  of  water,  sulphuric  acid,  and  other  bodies. 
The  volume  of  expired  carbon  dioxide  is  regularly  less  than  the  volume  of 

CO 
the  inspired  oxygen,  and  the  relation  -j-1 ,  which  is  called  the  respiratory 

quotient,  is  generally  less  than  1. 

1  In  regard  to  the  methods  for  estimating  the  carbon-dioxide  excretion  and  the 
oxygen  consumption,  see  Zuntz,  Hermann's  Handbuch  d.  Physiol,  4,  Tl.  2;  Hoppe- 
Seyler,  Zeitschr.  f.  physiol.  Chem.,  19;  Sonden  and  Tigerstedt,  Skand.  Arch.  f.  Physiol., 
6;  Speck,  Physiol,  des  menschl.  Atmens.  Leipzig,  1892;  Zuntz  and  Geppert,  Pfliiger's 
Arch.,  42;  Magnus-Levy,  ibid.,  55,  10,  where  the  works  of  Zuntz  and  his  pupils  are 
cited;  Hanriot  et  Richet,  Compt.  rend.,  104,  and  Atwater,  Bull,  of  Dept.  of  Agric, 
Washington,  Nos.  44,  03,  09,  and  109. 


DETERMINATION  OF   THE   EXTENT  OF  METABOLISM.  023 

The  magnitude  of  the  respiratory  quotient  is  dependent  upon  the  kind 
of  substances  destroyed  in  the  body.  In  the  combustion  of  pure  carbon 
one  volume  of  oxygen  yields  one  volume  of  carbon  dioxide,  and  the  quo- 
tient Is  therefore  equal  to  1.  The  same  is  true  in  the  burning  of  carbo- 
hydrates, and  in  the  exclusive  decomposition  of  carbohydrates  in  the 
animal  body  the  respiratory  quotient  must  be  approximately  1.  In  the  ex- 
clusive metabolism  of  proteids  it  is  close  to  0.80,  and  with  the  decomposi- 
tion of  fat  it  is  0.7.  In  starvation,  as  the  animal  draws  on  its  own  flesh 
and  fat,  the  respiratory  quotient  must  be  a  close  approach  to  the  Latter 
{'mure.  The  respiratory  quotient  therefore  gives  important  data  on  the 
quality  of  the  material  decomposed  in  the  body,  naturally  with  the  suppo- 
sition that  the  elimination  of  carbon  dioxide,  independent  of  the  formation 
of  carbon  dioxide,  is  not  influenced  by  special  conditions,  such  as  the  alter- 
ation of  the  respiratory  mechanism. 

It  is  also  possible  in  systematized  experimentation  to  carry  on  the 
metabolism  experiments  so  that  the  decomposable  material  of  the  body, 
as  shown  by  the  respiratory  quotient,  remains  qualitatively  the  same,  at 
least  for  a  short  time.  In  such  experiments  it  has  been  shown,  especially 
by  Zuntz  and  his  pupils,1  that  the  extent  of  oxygen  consumption  may 
be  taken  as  a  measure  for  the  action  of  different  influences  on  the  extent  of 
metabolism.  This  possibility  is  based  on  the  fact  proved  by  Pfluger  and 
his  pupils,  and  by  Voit,2  that  the  consumption  of  oxygen  within  wide 
limits  is  independent  of  the  supply  of  oxygen,  and  is  exclusively  dependent 
upon  the  oxygen  demand  of  the  tissues.  For  certain  reasons  the  consump- 
tion of  oxygen  gives  indeed  a  better  conclusion  than  the  elimination  of 
carbon  dioxide  as  to  the  extent  of  exchange  of  material  and  energy;  but 
as  the  same  quantity  of  oxygen  (100  grams)  consumes  different  quantities 
of  fat,  carbohydrates,  and  proteids  in  the  body — namely,  35,  84.4,  and 
74.4  grains  respectively — the  respiratory  quotient  must  also  be  deter- 
mined, in  order  to  ascertain  the  nature  of  the  substance  burnt  in  the  body, 
simultaneously  with  the  determination  of  the  carbon  dioxide. 

As  the  different  foods  require  different  amounts  of  oxygen  in  the  com- 
bustion of  each  gram  of  substance  and  yield  different  amounts  of  ('< ).,,  each 
gram  of  oxygen  taken  up  and  each  gram  of  carbon  in  the  expired  air  as 
carbon  dioxide  must  correspond  to  different  heat  values.  This  follows 
from  the  following  table: 

Calories 

per  gnu.  ( ' 

in  the  Ol  >.■  "f 

the  Expired  Air. 

In  the  combustion  of  cane-sugar.  ...     9.5 

"     "  "  "  meat 10.2 

"    "  "  "  fat 12.3 

'See  foot-note  1,  page  622. 

1  Pfluger,  Pfluger 's  Arch.,  6,  10,  and  14;  Finkler,  ibid.,  10;  Finkler  and  Oertmann» 
ibid.,  14;  Voit,  Zeitschr.  f.  Biologie,  11  and  14. 


Relative 
Value. 

Calories 
per  grm. 
(on -umed 
Oxygen. 

Relative 
Value. 

100 

3.56 

118.6 

107 

3.00 

100.0 

129 

3.27 

109.0 

62  i  METABOLISM  WITH  VARIOUS  FOODS. 

Pfluger  has  found  the  following  figures  for  the  calorific  value  of  1  gram 
oxygen: 

For  muscle  tissue  free  from  fat 3 .  30  Cal. 

Fat 3.29    " 

Starch 3.53    " 

The  figures  for  the  oxygen  differ,  as  seen  above,  less  than  tho  e  for  the  carbon, 
and  this  is  the  reason  why,  as  above  stated,  the  oxygen  consumption  gives  a  much 
more  correct  conclusion  as  to  the  exchange  of  force  than  the  elimination  of  carbon 
dioxide.1 

Kaufmann  2  encloses  the  individual  to  be  experimented  upon  in  a 
capacious  tin  box,  which  serves  both  as  a  respiration-chamber  and  a  calorim- 
eter, and  which  permits  of  the  estimation  of  the  nitrogen  of  the  urine  and 
the  carbon  dioxide  expired,  as  well  as  the  inspired  oxygen  and  the  quantity 
of  heat  produced.  If  we  start  from  the  theoretically  calculated  formulae 
for  the  various  possible  transformations  of  the  proteids,  fats,  and  carbo- 
hydrates in  the  body,  it  is  clear  that  other  values  must  be  obtained  for 
the  heat,  carbon  dioxide,  oxygen,  and  nitrogen  of  the  urine,  when  one,  for 
example,  admits  of  a  complete  combustion  of  proteids  to  urea,  carbon 
dioxide,  and  water,  or  of  a  partial  splitting  off  of  fat.  Another  relation- 
ship between  heat,  carbon  dioxide,  and  oxygen  is  also  to  be  expected  when 
the  fat  is  completely  burnt  or  when  it  is  decomposed  into  sugar,  carbon 
dioxide,  and  water.  In  this  way,  by  a  comparison  of  the  values  found  in 
special  cases  with  the  figures  calculated  for  the  various  transformations, 
Kaufmann  attempts  to  explain  the  various  decomposition  processes  in 
the  body  under  different  nutritive  conditions. 

I.  The  Energy  and  the  Relative  Nutritive  Value  of  Various  Organic 

Foodstuffs. 

With  the  organic  foods  the  organism  receives  a  supply  of  chemical 
energy  which  is  converted  into  heat  and  mechanical  work  in  the  body. 
This  energy  of  the  various  foods  may  be  represented  by  the  amount  of  heat 
which  is  set  free  in  their  combustion.  This  quantity  of  heat  is  expressed  as 
calories,  and  a  small  calorie  is  the  quantity  of  heat  necessary  to  warm  1  gram 
of  water  from  0°  to  1°  C.  A  large  calorie  is  the  quantity  of  heat  necessary  to 
warm  1  kilo  of  water  1°  C.  Here  and  in  the  following  pages  large  calories  are 
to  be  understood.  There  are  numerous  investigations  by  different  experi- 
menters, such  as  Frankland,  Danilewski,  Rurner,  Berthelot,  Stohmann, 
and  others,  on  the  calorific  value  of  different  foodstuffs.  The  following  re- 
sults, which  represent  the  calorific  value  of  a  few  nutritive  bodies  on  com- 
plete combustion  outside  of  the  body  to  the  highest  oxidation  products, 
are  taken  from  Stohmann  's  3  work. 

1  See  Ad.  Magnus-Levy,  Pfluger's  Arch.,  55,  7,  and  Pfluger,  ibid.,  77,  78,  and  79. 

2  Arch.  d.  Physiologie  (5),  8. 

3  See  Rubner,  Zeitschr.  f.  Biologie,  21,  which  also  cites  the  works  of  Frankland 
and  Danilewski;  see  also  Berthelot,  Compt.  rend.,  102,  104,  and  110;  Stohmann, 
Zeitschr.  f.  Biologie,  31. 


CALORIFIC   VALUE  OF  THE  FOODSTUFFS.  625 

Caloriea. 

Casein 5 .  86 

Ovalbumin 5.74 

Conglutin 5.48 

Proteid  (average) 5.71 

Animal  tissue-fat 9 .  50 

Butter-fat 9 .  23 

Cane-sugar 3 .  96 

Milk-sugar 3 .  95 

Dextrose 3 .  74 

Starch 4.19 

Fats  and  carbohydrates  are  completely  burnt  in  the  body,  and  one  can 
therefore  consider  their  combustion  equivalent  as  a  measure  of  the  living 
force  developed  by  them  within  the  organism.  We  generally  designate  9.3 
and  4.1  calories  for  each  gram  of  substance  as  the  average  for  the  physio- 
logical calorific  value  of  fats  and  carbohydrates  respectively. 

The  proteids  act  differently  from  the  fats  and  carbohydrates.  They 
are  only  incompletely  burnt,  and  they  yield  certain  decomposition  prod- 
ucts, which,  leaving  the  body  with  the  excreta,  still  represent  a  certain 
quantity  of  energy  which  is  lost  to  the  body.  The  heat  of  combustion  of 
the  proteids  is  smaller  within  the  organism  than  outside  of  it,  and  they 
must  therefore  be  specially  determined.  For  this  purpose  Rubner  l  fed  a 
dog  on  washed  meat,  and  he  subtracted  from  the  heat  of  combustion  of 
the  food  the  heat  of  combustion  of  the  urine  and  faeces,  which  corresponded 
to  the  food  taken  plus  the  quantity  of  heat  necessary  for  the  swelling  up  of 
the  proteids  and  the  solution  of  the  urea.  Rubner  has  also  tried  to  deter- 
mine the  heat  of  combustion  of  the  proteids  (muscle-proteids)  decomposed 
in  the  body  of  rabbits  in  starvation.  According  to  these  investigations, 
the  physiological  heat  of  combustion  in  calories  for  each  gram  of  substance 
is  as  follows : 

1  grm.  of  the  dry  substance.  Calories. 

Proteid  from  meat 4.4 

Muscle 4.0 

Proteid  in  starvation 3.8 

Fat  (average  for  various  fats) 9.3 

Carbohydrates  (calculated  average) 4.1 

The  physiological  combustion  value  of  the  various  foods  belonging  to 
the  same  group  is  not  quite  the  same.  It  is,  for  instance,  3.97  calories  for 
a  vegetable  proteid,  conglutin,  and  4.42  calories  for  an  animal  proteid  body, 
syntonin.  According  to  Rubner  the  normal  heat  value  per  1  gram  of 
animal  proteid  may  be  considered  as  4.23  calories,  and  of  vegetable  proteid 
as  3.96  calories.  When  a  person  on  a  mixed  diet  takes  about  60  per  cent  of 
the  proteids  from  animal  foods  and  about  40  per  cent  from  vegetable  foods, 
the  value  of  1  gram  of  the  proteid  of  the  food  is  equivalent  to  about 
4.1  calories.     The  physiological  value  of  each  of  the  three  chief  groups  of 

1  Zeitschr.  f.  Biologie,  21. 


626  METABOLISM  WITH   VARIOUS  FOODS. 

organic  foods,  by  their  decomposition  in  the  body,  is  in  round  numbers  as 
follows : 

Calories. 

1  gram  proteid 4.1 

1     "     fat 9.3 

1     ' '     carbohydrate 4.1 

As  will  be  shown,  the  fats  and  carbohydrates  may  decrease  the  metab- 
olism of  proteids  in  the  body,  while,  on  the  other  hand,  the  quantity  of 
proteids  in  the  body  or  in  the  food  acts  on  the  metabolism  of  fat  in  the 
body.  In  physiological  combustion  the  various  foods  may  replace  one 
another  to  a  certain  extent,  and  it  is  therefore  important  to  know  the 
ratio  of  replacement.  The  investigations  made  by  Rubner  have  taught 
that  this,  if  it  relates  to  the  force  and  heat  production  in  the  animal  body, 
is  a  proportion  that  corresponds  with  the  figures  of  the  heat  value  of  the 
same.  This  is  apparent  from  the  following  table.  In  this  is  found  the 
weight  of  the  various  foods  equal  to  100  grams  of  fat,  a  part  determined  from 
experiments  on  animals  and  a  part  calculated  from  figures  of  the  heat  values. 

100  grams  fat  are  equal  to  or  isodynamic  with 

From  Experiments  From  the  Difference, 

on  Animals.  Heat  Value.  per  cent. 

Syntonin 225  213  +5.6 

Muscle-flesh  (dried) 243  235  +4.3 

Starch 232  229  +1.3 

Cane-sugar... 234  235  -0 

Dextrose 256  255  -0 

From  the  given  isodynamic  value  of  the  various  foods  it  follows  that 
these  substances  replace  one  another  in  the  body  almost  in  exact  ratio  to 
the  energy  contained  in  them.  Thus  in  round  numbers  227  grams  of  pro- 
teid and  carbohydrate  are  equal  to  or  isodynamic  with  100  grams  of  fat  in 
regard  to  source  of  energy,  because  each  yields  930  calories  on  combustion 
in  the  body. 

By  means  of  recent  very  important  calorimetric  investigations  Rubner  * 
has  shown  that  the  heat  produced  in  an  animal  in  several  series  of  experi- 
ments extending  over  forty-five  days  corresponded  to  within  0.47  per  cent 
of  the  physiological  heat  of  combustion  calculated  from  the  decomposed 
body  and  foods.  Atwater  and  his  collaborators  2  have  made  some  very 
thorough  investigations  on  this  subject  on  men.  In  their  experiments 
they  made  use  of  a  large  respiration  calorimeter,  which  not  only  deter- 
mined exactly  the  excreta  but  also  made  a  calorimetric  determination  of 
the  heat  given  out  by  the  person  experimented  upon,  i.e.,  the  work  per- 
formed. From  the  results  of  these  experiments  they  found  nearly  an 
absolutely  complete  agreement  between  the  calories  found  directly  and 
those  calculated. 

1  Zeitschr.  f.  Biologie,  30. 

2  Bull,  of  Dept.  of  Agric,  Washington,  44,  03,  G9,  and  109. 


BEAT   VALUE  OF  THE  Food.  627 

This  isodynamio  law  is  of  fundamental  value  in  the  study  of  metab- 
olism and  nutrition.  By  th's  law  it  is  possible  to  consider  the  pro© 
of  metabolism  as  more  uniform  transformations  of  energy.  The  quantity 
of  energy  in  the  transformed  foods  or  the  constituents  of  the  body  may 
be  used  as  a  measure  for  the  total  consumption  of  energy,  and  the 
knowledge  of  the  quantity  of  energy  in  the  foods  must  also  be  the  basis  for 
the  calculation  of  dietaries  for  human  beings  under  various  conditions. 

The  heat  value  of  a  foodstuff  can  be  directly  determined  in  a  calorim- 
eter but  may  also  be  calculated  from  its  composition.  If  one  subtracts 
from  the  gross  heat  value  of  the  food  obtained  in  one  way  or  another,  the 
combustion  heat  of  the  faeces  and  urine  with  the  same  diet,  there  is  obtained 
the  net  calorific  value  of  the  diet.  This  value,  calculated  in  percentage  of 
the  total  energy  content  of  the  food,  is  called  the  physiological  availability 
by  RtJBNER.1  In  order  to  elucidate  this  we  will  give  a  few  of  Rubner's 
values.  The  loss  in  calories,  as  well  as  the  physiological  availability,  are 
calculated  in  percentages  of  the  total  energy  content  of  the  food. 

p     j                                                Loss  in  per  cent.  Total  loss  Availability 

In  urine.  In  the  faeces,     in  per  cent,  in  per  cent. 

Cow's  milk 5. 13  5.07  10.20  s<)  s 

Mixed  diet  (rich  in  fat) 3.87  5.73  9.60  90.4 

"     (poorinfat) 4.70  6.00  10.70  89.3 

Potatoes 2.00  5.60  7.60  92.4 

Graham  bread 2.40  15.50  17.90  82.1 

Rye  bread 2.20  24.30  26.50  73  5 

Meat 16.30  6.90  23.20  76.8 

In  order  to  simplify  the  calculation  of  the  energy  exchange  there  exists, 

besides  the  above-mentioned  standard  figuras  for  the  physiological  calorific 

value  of  the  organic  foodstuffs,  also  for  the  carbon  of  the  carbon  dioxide, 

and    for   the   oxygen   other   standard  factors.     Thus  for  1   gram  of   meat 

(dry  substance)    free  from   fat   and  extractives  we   have  the  calculated 

value   of   5.44-5.77  Cal.     Kohler  2  found    5.678  Cal.    for  1  gram  of  ash 

and   fat-free    dried-meat    substance  of   the    ox    and    5.599    Cal.    for  the 

horse.     According  to    Frentzel    and    Schreuer  3    45.4    Cal.    i  s    calcu- 

lated    for    1    gram    of   nitrogen    in   fat   and    ash-free    dried-meat    faeces 

(dog),  while  6.97  to  7.45  Cal.   is  calculated  for   1  gram  of   nitrogen  in 

meat-urine.      The  calorific  urine  quotient  -^r1  seems  still,  as  found  by 

Tangl,  not  to  be  constant  for  human  beings  at  least,  but  is  dependent 
upon  the  variety  of  food. 

Instead  of  the  direct  determination  the  heat  of  combustion  can  also  bo  deter- 
mined from  the  elementary  composition  according  to  the  following  principle  as 
suggested  by  E.  Voit.4     If  we  designate  the  heat  of  combustion  for  1  gram  of 

1  Zeitschr.  f.  Biologie,  42. 

2  Zeitschr.  f.  physiol.  ('hem.,  31. 

3  The  works  of  Frentzel  and  Schreuer  may  be  found  in  Arch.  f.  (Anat.  u.)  Physiol 
1901,   1902,  and  1903. 

*  Zeitschr.  f.  Biologie,  44.     See  also  Krummacher,  ibid. 


628  METABOLISM  WITH   VARIOUS  FOODS. 

the  substance  by  Cal.  and  the  quantity  of  oxygen  necessary  for  the  complete 
combustion  of  1  gram  of  the  substance  ( =  oxygen  capacity  of  the  substance)  by 

0,  then  -~  =  K,  which  i  the  combustion  value  for  1  gram    f  oxygen.     The  oxygen 

capacity  can  be  calculated  from  the  elementary  composition,  and  when  the  value 
of  K  is  known,  the  combustion  heat  of  a  chemical  compound  or  a  known  mixture 
can  be  readi  y  determined.  The  value  K  is  nearly  constant  or  substances  of 
the  same  groups;  but  also  different  groups  show  among  themselves  only  slight 
deviation  for  this  value.  Voit  obtained  the  following  values  for  a  few  of 
the  foodstuffs: 

K  (in  kg.  Cal.).  O  Capacity. 

Plant  proteid 3 .  298  1 .  740 

Animal  proteid 3.273  1.741 

Fat 3.271  2.863 

Carbohydrate 3 .  525  1 .  156 

These  methods  of  calculation  are,  according  to  Voit  and  Krummacher,  admis- 
sible for  practical  purposes. 

II.   Metabolism  in  Starvation. 

In  starvation  the  decomposition  in  the  body  continues  uninterruptedly , 
though  with  decreased  intensity;  but,  as  it  takes  place  at  the  expense  of 
the  substance  of  the  body,  it  can  only  continue  for  a  limited  time.  When 
an  animal  has  lost  a  certain  fraction  of  the  mass  of  the  body  death  is  the 
result.  This  fraction  varies  with  the  condition  of  the  body  at  the  begin- 
ning of  the  starvation  period.  Fat  animals  succumb  when  the  weight  of 
the  body  has  sunk  to  one  half  of  the  original  weight.  Otherwise,  accord- 
ing to  Chossat,1  animals  die  as  a  rule  when  the  weight  of  the  body  has 
sunk  to  two  fifths  of  the  original  wreight.  The  period  when  death  occurs 
from  starvation  not  only  varies  with  the  varied  nutritive  condition  at  the 
beginning  of  starvation,  but  also  with  the  more  or  less  active  exchange  of 
material.  This  is  more  active  in  small  and  young  animals  than  in  large 
and  older  ones,  but  different  classes  of  animals  show  an  unequal  activity. 
Children  succumb  in  starvation  in  3-5  days  after  having  lost  one  fourth 
of  their  body  mass.  Grown  persons,  as  observed  on  Succr,2  may  starve 
for  twenty  days  without  lasting  injury ;  and  there  are  reports  of  cases  of 
starvation  extending  over  a  period  of  even  more  than  fifty  days.  Dogs 
can  live  without  food  from  four  to  eight  weeks,  birds  five  to  twenty  days, 
snakes  more  than  half  a  year,  and  frogs  more  than  a  year. 

In  starvation  the  weight  of  the  body  decreases.  The  loss  of  weight  is- 
greatest  in  the  first  few  days,  and  then  decreases  rather  uniformly.  In 
small  animals  the  absolute  loss  of  weight  per  day  is  naturally  less  than 
in  larger  animals.  The  relative  loss  of  weight — that  is,  the  loss  of  weight 
of  the  unit  of  the  weight  of  the  body,  namely,  1  kilo — is,  on  the  contrary, 
greater  in  small  animals  than  in  larger  ones.     The  reason  for  this  is  that 

1  Cited  from  Voit  in  Hermann's  Handbuch,  6,  Thl.  1,  100. 

2  See  Luciani,  Das  Hungern.     Hamburg  u.  Leipzig,  1890. 


METABOLISM   IN  STARVATION.  629 

the  smaller  animate  have  a  greater  surface  of  body  in  proportion  to  their 
mass  than  larger  animals,  and  the  greater  loss  of  heat  caused  thereby  must 
be  replaced  by  a  more  aethre  consumption  of  material. 

It  follows  from  the  decrease  in  the  weight  of  the  body  that  the  absolute 
extent  of  metabolism  must  diminish  in  starvation.  If,  on  the  contrary, 
the  extent  of  the  metabolism  is  leferred  to  the  unit  of  the  weight  of  the 
body,  namely.  1  kilo.it  appears  that  this  quantity  remains  nearly  unchanged 
during  starvation.  The  investigations  of  Zuxtz,  Lkhmaw,  and  others  ' 
on  Cktti  showed  on  the  third  and  sixth  days  of  starvation  an  average 
consumption  of  4.65  c.  c.  oxygen  per  kilo  in  one  minute,  and  on  the  ninth 
to  eleventh  day  an  average  of  4.73  c.  c.  The  calories,  as  a  measure  of  the 
metabolism,  fell  on  the  first  to  fifth  day  of  starvation  from  1850  to  1600 
calories,  or  from  32.4  to  30  per  kilo,  and  it  remained  nearly  unchanged, 
if  referred  to  the  unit  of  body  weight.2 

The  extent  of  the  metabolism  of  proteids,  or  the  elimination  of  nitrogen 
by  the  urine,  which  is  a  measure  of  the  same,  diminishes  as  the  weight  of 
the  body  diminishes.  This  decrease  is  not  regular  or  the  same  during 
the  entire  period  of  starvation,  and  the  extent  depends,  as  the  experiments 
made  upon  carnivora  have  shown,  upon  several  circumstances.  During 
the  first  few  days  of  starvation  the  excretion  of  nitrogen  is  greatest,  and 
the  richer  the  body  is  in  proteid,  due  to  the  food  previously  taken,  the 
greater  is  the  proteid  catabolism  or  the  nitrogen  elimination,  according 
to  Voir.  The  nitrogen  elimination  diminishes  the  more  rapidly — that  is, 
the  curve  of  the  decrease  is  more  sudden — the  richer  in  proteids  the  food 
was  which  was  taken  before  starvation.  This  condition  is  apparent  from 
the  following  table  of  data  of  three  different  starvation  experiments  made 
by  Vorr  3  on  the  same  dog.  This  dog  received  2500  grams  of  meat  daily 
before  the  first  series  of  experiments,  1500  grams  of  meat  daily  before 
the  second  series,  and  a  mixed  diet  relatively  poor  in  nitrogen  before  the 
third  series. 

Dav  of  Starvation  Grams  of  Urea  Eliminated  in  Twenty-four  Hours. 

_  Ser.  I.  Ser.  II.  Ser.  III. 

First 60.1  26.5  13.8 

Second 24.9  18.6  115 

Third 19.1  15.7  10  2 

Fourth 17.3  14.9  12  2 

Fifth 12.3  14.8  12  1 

Sixth 13.3  12.8  12  6 

Seventh 12.5  12.9  11  ;? 

Eighth 10.1  12.1  10.7 

In  man  and  also  in  animals  sometimes  a  rise  in  the  nitrogen  excretion 
is  observed  about  the  second  or  third  starvation  day.  which  is  then  fol- 

1  Berlin,  klin.  Wochenschr.,  1887. 

2  See  also  Tigerstedt  and  collaborators  in  Skand.  Arch.  f.  Physiol.,  7. 

3  See  Hermann's  Handbuch,  6,  Thl.    1,  S9. 


630  METABOLISM  WITH   VARIOUS  FOODS. 

lowed  by  a  regular  diminution.  This  rise  is  explained  by  Prausxitz, 
Tigf.rstedt,  Laxdergrex,1  as  f ollows :  At  the  commencement  of  star- 
vation the  proteid  metabolism  is  reduced  by  the  glycogen  still  present 
in  the  body.  After  the  consumption  of  the  glycogen,  which  takes  place  in 
great  part  during  the  first  days  of  starvation,  the  destruction  of  proteids 
increases  as  the  glycogen  action  decreases,  and  then  decreases  again  when 
the  body  has  become  poorer  in  available  proteids. 

Other  conditions,  such  as  varying  quantities  of  fat  in  the  body,  have 
an  influence  on  the  rapidity  with  which  the  nitrogen  is  eliminated  during 
the  first  days  of  starvation.  After  the  first  few  days  of  starvation  the 
elimination  of  nitrogen  is  more  uniform.  It  may  diminish  gradually  and 
regularly  until  the  death  of  the  animals  or  there  may  be  a  rise  in  the  last 
days,  a  so-called  ante-mortem  increase.  Whether  the  one  or  the  other 
occurs  depends  upon  the  relationship  between  the  proteid  and  fat  content 
of  the  body. 

Like  the  destruction  of  proteids  during  starvation,  the  decomposition 
of  fat  proceeds  uninterruptedly  and  the  greatest  part  of  the  calories  needed 
during  starvation  are  supplied  by  the  fats.  According  to  Rubxer  and 
Voit  the  proteid  catabolism  varies  only  slightly  in  starving  animals  at 
rest  and  at  an  average  temperature,  and  forms  a  constant  portion  of  the 
total  exchange  of  energy;  of  the  total  calories  in  dogs  10-16  per  cent  comes 
from  the  proteid  decomposition  and  84-90  per  cent  from  the  fats.  This  is 
at  least  true  for  starving  animals  which  had  a  sufficiently  great  original 
fat  content.  If  on  account  of  starvation  the  animal  has  become  relatively 
poorer  in  fat  and  the  fat  content  of  the  body  has  fallen  below  a  certain 
limit,  then  in  order  to  supply  the  calories  necessary  a  larger  quantity  of 
proteid  is  destroyed  and  the  ante-mortem  increase  now  occurs  (E.  Voit  2). 

Since  the  fat  has  a  diminishing  influence  on  the  destruction  of  proteids 
corresponding  to  what  was  said  above,  the  elimination  of  nitrogen  in  star- 
vation is  less  in  fat  than  in  lean  individuals.  For  instance,  only  9  grams 
of  urea  were  voided  in  twenty-four  hours  during  the  later  stages  of  starva- 
tion by  a  well-nourished  and  fat  person  suffering  from  disease  of  the  brain, 
while  I.  Muxk  found  that  20-29  grams  urea  were  voided  daily  by  Cetti,3 
who  had  been  poorly  nourished. 

The  investigations  on  the  exchange  of  gas  in  starvation  have  shown,  as 
previously  mentioned,  that  the  absolute  extent  of  the  same  is  diminished, 
but  that  when  the  consumption  of  oxygen  and  elimination  of  carbon  diox- 

1  Prausnitz,  Zeitschr.  f.  Biologie,  29;  Tigerstedt  and  collaborators,  1.  c. ;  Lander- 
gren,  "Undersokningar  ofver  menniskans  agghviteomsiittning,  Inaug.-Diss.  Stock- 
holm, 1902. 

2  Zeitschr.  f.  Biologie,  41,  167  and  502.  See  also  Kaufmann,  ibid,  and  N.  Schulz. 
ibid.,  and  Pfliiger's  Arch.,  76. 

3  Berl.  klin.  Wochenschr.,  1887. 


METABOLISM  IN  STARVATION.  631 

ide  are  calculated  on  the  unit  of  weight  of  the  body,  1  kilo,  this  quantity 
quickly  sinks  to  a  minimum  and  then  remains  unchanged,  or,  oil  the  con- 
tinuation of  the  starvation,  may  actually  rise.  It  is  a  well  known 
fact  that  the  body  temperature  of  starving  animals  remains  nearly  con- 
stant, without  showing  any  appreciable  decrease,  during  the  greater  part 
of  the  starvation  period.  The  temperature  of  the  animal  first  sinks  a  few 
days  before  death,  and  death  occurs  at  about  33-30°  C. 

From  what  has  been  said  about  the  respiratory  quotient  it  follows  that 
in  starvation  it  is  about  the  same  as  with  fat  and  meat  exclusively  as  food, 
i.e.,  approximately  0.7.  This  is  often  the  case,  but  it  may  occasionally  be 
lower,  0.65-0.50,  as  observed  in  the  cases  of  Cetti  and  Succi.  As  explana- 
tion for  this  unexpected  behavior  one  must  admit  of  a  storage  of  incom- 
pletely oxidized  substances  in  the  body  during  starvation. 

Water  passes  uninterruptedly  from  the  body  in  starvation  even  when 
none  is  taken.  If  the  quantity  of  water  in  the  tissues  rich  in  proteids  is 
considered  as  70-80  per  cent,  and  the  quantity  of  proteids  in  the  same 
20  per  cent,  then  for  each  gram  of  proteid  destroyed  about  4  grams  of 
water  is  set  free.  This  liberation  of  water  from  the  tissues  is  generally 
sufficient  to  supply  the  loss  of  water  and  starvation  is  ordinarily  not  accom- 
panied with  thirst.     Starving  animals,  as  a  rule,  do  not  partake  of  water. 

The  loss  of  water  calculated  on  the  percentage  of  the  total  organism  must 
naturally  be  essentially  dependent  upon  the  previous  amount  of  fatty  tissue  in 
the  body.  If  we  bear  these  conditions  in  mind,  then  it  seems,  according  to  Boht- 
i.inok.,1  that,  from  experiments  upon  white  mice,  the  animal  body  is  poorer  in 
water  during  inanition.  The  body  loses  more  water  than  is  set  free  by  the  de- 
struction of  the  tissues. 

The  mineral  substances  leave  the  body  uninterruptedly  in  starvation 
until  death,  and  the  influence  of  the  destruction  of  tissues  is  plainly  per- 
ceptible by  their  elimination.  Because  of  the  destruction  of  tissues  rich  in 
potassium  the  proportion  between  potassium  and  sodium  in  the  urine 
changes  in  starvation,  so  that,  contrary  to  the  normal  conditions,  the 
potassium  Is  eliminated  in  proportionately  greater  quantities.  Munk  also 
observed  in  Cetti 's2  case  a  relative  increase  in  the  phosphoric  acid  and 
calcium  in  the  urine  during  starvation,  which  was  due  to  an  increased 
exchange  of  bone-substance. 

Contrary  to  the  above  Bohtlingk  with  starving  white  mice  and  Katsutama  3 
with  starving  rabbits  found  a  greater  excretion  of  sodium  than  potassium. 

The  question  as  to  the  participation  of  the  different  organs  in  the  loss 
of  weight  of  the  body  during  starvation  is  of  special  interest.     In  elucida- 

1  Arch,  des  sciences  biol.  de  St.  Petersbourg,  5. 

2L.  c. 

5  Bohtlingk,  1.  c. ;   Katsuyama,  Zeitschr.  f.  physiol.  Chem.,  26. 


632  METABOLISM  WITH   VARIOUS  FOODS. 

tion  of  this  point  we  give  the  following  results  of  Chossat's  experiments 
on  pigeons,  and  those  of  Voit  1  on  a  male  cat.  The  results  are  percentages 
of  weight  lost  from  the  original  weight  of  the  organ. 

Pigeon  (Chossat).      Male  Cat  (Voit). 

Adipose  tissue 93  per  cent.  97  per  cent. 

Spleen 71    "  "  67   "  " 

Pancreas 64   "  "  17   "  " 

Liver 52"  "  54"  " 

Heart 45   "  "  3   "  " 

Intestine 42"  "  18"  " 

Muscles 42   "  "  31    "  " 

Testicles —  40   "  " 

Skin 33   "  "  21   '  [  " 

Kidneys 32   "  ' «  26   "  " 

Lungs 22   "  "  18   "  " 

Bones 17   "  "  14  "  " 

Nervous  system 2   "  "  3   "  " 

The  total  quantity  of  blood,  as  well  as  the  quantity  of  solids  contained 
therein,  decreases,  as  Pantjm  and  others2  have  shown,  in  the  same  propor- 
tion as  the  weight  of  the  body.  The  statements  in  regard  to  the  loss 
of  water  by  different  organs  are  somewhat  contradictory;  according  to 
Lukjaxow  3  it  seems  that  the  various  organs  act  somewhat  differently  in 
this  respect. 

The  above-tabulated  results  cannot  serve  as  a  measure  of  the  metabo- 
lism in  the  various  organs  during  starvation.  For  instance,  the  nervous 
system  shows  only  a  small  loss  of  weight  as  compared  with  the  other  organs, 
but  from  this  it  must  not  be  concluded  that  the  exchange  of  material  in  this 
system  of  organs  is  least  active.  The  condition  may  be  quite  different; 
for  one  organ  may  derive  its  nutriment  during  starvation  from  some  other 
organ  and  exist  at  its  expense.  A  positive  conclusion  cannot  be  drawn  in 
regard  to  the  activity  of  the  metabolism  in  an  organ  from  the  loss  of  weight 
of  that  organ  in  starvation.  Death  by  starvation  is  not  the  result  of  the  death 
of  all  the  organs  of  the  body,  but  it  depends  more  likely  upon  the  disturb- 
ance in  the  nutrition  of  a  few  less  vitally  important  organs  (E.  Voit  4). 

The  knowledge  of  metabolism  during  starvation  is  of  the  greatest  im- 
portance in  the  study  of  nutrition,  and  it  forms  to  a  certain  extent  the 
starting-point  for  the  study  of  metabolism  under  different  physiological  and 
pathological  conditions.  To  answer  the  question  whether  the  metabolism 
of  a  person  in  a  special  case  is  abnormally  increased  or  diminished  it  is 
naturally  very  important  to  know  the  average  extent  of  metabolism  of  a 
healthy  person  under  the  same  circumstances,  for  comparison.  This  quan- 
tity can  be  called  the  abstinent  value,  that  is,  the  extent  of  metabolism 
used  in  absolute  bodily  rest  and  inactivity  of  the  intestinal  tract.     As 

1  Cited  from  Voit  in  Hermann's  Handbuch,  (>,  Part  I,  96  and  97. 

2  Panum,  Virchow's  Arch.,  29;  London,  Arch.  d.  scienc.  biol.  de  St.  P£tersbourg,  4. 

3  Zeitschr.  f.  physiol.  Chem.,  13. 

4  Zeitschr.  f.  Biologie,  41. 


METABOLISM    WITH  INADEQUATE  NUTRITION.  633 

a  measure  of  this  quantity  we  determine,  according  to  Geppert-Zuntz,  the 
extent  of  gaseous  exchange,  and  especially  the  consumptioD  of  oxygen,  of  a 
person  lying  down,  best  sleeping,  in  the  early  morning  and  at  least  twelve 
hours  after  a  light  meal  not  rich  in  carbohydrates.  The  gas  volume 
reduced  to  0°  C.  and  7(i()  mm.  Hg  pressure  is  calculated  on  1  kilo  of  body 
weight  and  for  one  minute  The  results  vary  between  3  and  4..">  for  the 
consumption  of  oxygen,  and  between  2.5  and  3.5  c.  c.  for  the  carbon  diox- 
ide. As  average  3.81  c.  c.  oxygen  and  3.08  c.  c.  carbon  dioxide  are  usu- 
ally given.1 

The  extent  of  proteid  destruction  cannot  be  determined  in  transient 
experiments,  and  for  these  reasons  only  the  values  found  after  several  days 
(if  starvation  are  useful.  In  the  starvation  experiments  on  Cetti  and 
Succi  the  elimination  of  nitrogen  per  kilo  on  the  fifth  to  the  tenth  starvation 
day  was  0.150-0.202  gram  N.  In  a  recent  starvation  experiment  made 
by  E.  and  0.  Freund  2  upon  Succi  the  nitrogen  excretion  on  the  twenty- 
first  day  sank  to  2.82  grams  N.  The  portion  of  the  urea  nitrogen  of  the 
total  nitrogen  sank  from  85-89  per  cent  on  the  first  days  of  starvation 
to  73  per  cent  on  the  fifteenth  day  and  56-54  per  cent  on  the  day  before 
the  last  day  of  starvation.  None  of  the  other  nitrogenous  constituents 
examined  increased  to  the  same  extent  as  the  urea  decreased.  The 
amount  of  neutral  sulphur  rose  from  10  to  40  per  cent  of  the  total  sulphur. 

III.   Metabolism  with  Inadequate  Nutrition. 

The  food  may  be  quantitatively  insufficient,  and  the  final  restdt  is 
absolute  inanition.  The  food  may  also  be  qualitatively  insufficient  or,  as 
we  say,  inadequate.  This  occurs  when  any  of  the  necessary  nutritive 
bodies  are  absent  in  the  food,  while  the  others  occur  in  sufficient  or  perhaps 
even  in  excessive  amounts. 

Lack  of  Water  in  the  Food.  The  quantity  of  water  in  the  organism  is 
greatest  during  fcetal  life,  and  then  decreases  with  increasing  age.  Natu- 
rally, the  quantity  differs  in  various  organs.  The  tissue  in  the  body  being 
poorest  in  water  is  the  enamel,  which  is  almost  free,  containing  only  2  p.  in. 
water,  the  teeth  about  100  p.  m.,  the  fatty  tissues  60-120  p.  m.  The  bones, 
with  140-440  p.  m.,  and  the  cartilage,  witli  540-740  p.  m.,  are  somewhat 
richer  in  water,  while  the  muscles,  blood,  and  glands,  with  750  to  more  than 
800  p.  m.,  are  still  richer.  The  quantity  of  water  is  even  greater  in  the 
animal  fluids  (see  preceding  chapter),  and  the  adult  body  contains  in  all 
about  630  p.  m.  water.3  If  it  is  borne  in  mind  that  two  thirds  of  the  animal 
organism  consists  of  water;  that  water  is  of  the  very  greatest  importance 
in  the  normal,  physical  composition  of  the  tissues;   moreover  that  all  flow 

1  See  v.  Noorden,  Lehrbuch  der  Pathologie  des  Stoffwechsel,  Berlin,  1893,  94. 

2  Wien.  klin.  Rundschau,  1901,  Nos.  5  and  6. 
sSee  Voit  in  Hermann's  Handbuch,  6,  Tl.  I,  345 


634  METABOLISM  WITH   VARIOUS  FOODS. 

of  juices,  all  exchange  of  substance,  all  supply  of  nutrition,  all  increase  or 
destruction,  and  all  discharge  of  the  products  of  destruction,  are  depend- 
ent upon  the  presence  of  water;  and,  in  addition,  that  by  its  evaporation 
it  is  an  important  regulator  of  the  temperature  of  the  body,  we  perceive 
that  water  must  be  necessary  for  life.  If  the  loss  of  water  be  not  replaced 
by  fresh  supplies  sooner  or  later,  the  organism  succumbs  and  death  may 
occur  earlier  with  lack  of  water  than  with  complete  inanition  (Landauer, 
Nothwang). 

If  the  water  is  abstracted  for  a  certain  time,  as  Landauer  and  especially 
W.  Straub  have  shown,  it  has  an  accelerating  influence  upon  the  decom- 
position of  proteid.  This  increased  metabolism  has,  according  to  Lan- 
dauer, the  purpose  of  replacing  a  part  of  the  water  abstracted  (because 
of  the  increased  metabolism).  The  abstraction  of  water  for  a  short  time 
may,  according  to  Spiegler,1  especially  in  man,  cause  a  diminution  in  the 
proteid  metabolism  by  means  of  a  reduced  proteid  absorption. 

Lack  of  Mineral  Substances  in  the  Food.  In  a.  previous  chapter  atten- 
tion was  called  in  several  instances  to  the  importance  of  the  mineral 
bodies  and  also  to  the  occurrence  of  certain  mineral  substances  in  certain 
amounts  in  the  various  organs.  The  mineral  content  of  the  tissues  and 
fluids  is  not  very  great  as  a  rule.  With  the  exception  of  the  skeleton, 
which  contains  about  220  p.  m.  mineral  bodies  (Volkmann  2),  the  animal 
fluids  or  tissues  are  poor  in  inorganic  constituents,  and  the  quantity  of 
such  amounts,  as  a  rule,  only  to  about  10  p.  m.  Of  the  total  quantity  of 
mineral  substances  in  the  organism,  the  greatest  part  occurs  in  the  skeleton, 
830  p.  m.,  and  the  next  greatest  in  the  muscles,  about  100  p.  m.  (Volk- 
mann). 

The  mineral  bodies  seem  to  be  partly  dissolved  in  the  fluids  and  partly 
combined  with  organic  substances.  In  accordance  with  this  the  organism 
persistently  retains,  with  food  poor  in  salts,  a  part  of  the  mineral  sub- 
stances, also  such  as  are  soluble,  as  the  chlorides.  On  the  burning  of  the 
organic  substances  the  mineral  bodies  combined  therewith  are  set  free  and 
may  be  eliminated.  It  is  also  admitted  that  they  in  part  combine  with 
the  new  products  of  the  combustion,  and  in  part  with  organic  nutritive 
bodies  poor  in  salts  or  nearly  salt-free,  which  are  absorbed  from  the  intes- 
tinal canal  and  are  thus  retained  (Voit,  Forster  3). 

If  this  statement  is  correct,  it  is  possible  that  a  constant  supply  of 
mineral  substances  with  the  food  is  not  absolutely  necessary,  and  that  the 
amount  of  inorganic  bodies  which  must  be  administered  is  insignificant. 

landauer,  Maly's  Jahresber.,  24;  Nothwang,  Arch.  f.  Hygiene,  1892;  Straub, 
Zeitschr.  f.  Biologie,  37  and  38;  Spiegler,  ibid.,  40. 

2  See  Voit  in  Hermann's  Handbuch,  6,  Part  1,  353. 

8  Forster,  Zeitschr.  f.  Biologie,  9.  See  also  Voit  in  Hermann's  Handbuch,  6, 
Part  1,  354. 


LACK  OF  MINERAL  SUBSTANCES  IN    THE  FOOD  635 

The  question  whether  this  be  so  or  not  has  not,  especially  in  man,  been 
sufficiently  investigated;  but  generally  we  consider  the  need  of  mineral 
substances  by  man  as  very  small.  It  may,  however,  be  assumed  that  man 
usually  takes  with  his  food  a  considerable  excess  of  mineral  substances. 

Experiments  to  determine  the  action  of  an  insufficient  supply  of  mineral 
substances  with  the  food  in  animals  have  been  made  by  several  investi- 
gators, especially  F0B8TER.  He  observed,  on  experimenting  with  dogs  and 
pigeons  with  food  as  poor  as  possible  in  mineral  substances,  that  a  very  sug- 
gestive  disturbance  of  the  functions  of  the  organs,  particularly  the  muscles 
and  the  nervous  system,  appeared,  and  that  death  resulted  in  a  short  time, 
earlier  in  fact  than  in  complete  starvation.  In  opposition  to  these  obser- 
vations Bunge  has  suggested  that  the  early  death  in  these  cases  was  not 
caused  by  the  lack  of  mineral  salts,  but  more  likely  by  the  lack  of  bases 
necessary  to  neutralize  the  sulphuric  acid  formed  in  the  combustion  of  the 
proteids  in  the  organism;  these  bases  must  then  be  taken  from  the  tissues. 
In  accordance  with  this  view,  Bunge  and  Lunin  !  also  found,  in  experiment- 
ing with  mice,  that  animals  which  received  nearly  ash-free  food  with  the 
addition  of  sodium  carbonate  were  kept  alive  twice  as  long  as  those  which 
had  the  same  food  without  the  sodium  carbonate.  Special  experiments  also 
show  that  the  carbonate  cannot  be  replaced  by  an  equivalent  amount  of 
sodium  chloride,  and  that  to  all  appearances  it  acts  by  combining  with  the 
acids  formed  in  the  body.  The  addition  of  alkali  carbonate  to  the  other- 
wise nearly  ash-free  food  may  indeed  delay  death,  but  cannot  prevent  it, 
and  even  in  the  presence  of  the  necessary  amount  of  bases  death  results 
for  lack  of  mineral  substances  in  the  food. 

In  the  above  series  of  experiments  made  by  Bunge  the  food  of  the 
animal  consisted  of  casein,  milk-fat,  and  cane-sugar.  While  milk  alone 
was  an  adequate  and  sufficient  food  for  the  animal,  Bunge  found  that  the 
animal  could  not  be  kept  alive  longer  by  food  consisting  of  the  above  con- 
stituents of  milk  and  cane-sugar  with  the  addition  of  all  the  mineral  sub- 
stances of  milk  than  with  the  food  mentioned  in  the  above  experiments 
with  the  addition  of  alkali  carbonate.  The  question  whether  this  result 
i<  to  be  explained  by  the  fact  that  the  mineral  bodies  of  milk  are  chem- 
ically combined  with  the  organic  constituents  of  the  same  and  can  be  assim- 
ilated only  in  such  combinations,  or  whether  it  depends  on  other  condi- 
tions, BuNGB  leaves  undecided.  These  observations,  however,  show  how 
difficult  it  is  to  draw  positive  conclusions  from  experiments  made  thus 
far  with  food  poor  in  salts.  Further  investigatioas  on  this  subject  seem 
to  be  necessary. 

With  an  insufficient  supply  of  chlorides  with  the  food  the  elimination 
of  chlorine    by  the  urine  decreases  constantly,  and  at  last  it    may 

'Bunge,  Lehrbuch  der  physiol.  Chem.,  4.  Aufl..  97;  Lunin,  Zeitschr.  f.  physio]. 
Chem  ,  .">. 


63b  METABOLISM   WITH  VARIOUS  FOODS. 

entirely,  while  the  tissues  still  persistently  retain  the  chlorides.  It  has 
already  been  stated  (Chapter  IX)  how  chloride  starvation  influences  other 
functions,  especially  the  secretion  of  gastric  juice.  If  there  be  a  lack  of 
sodium  as  compared  with  potassium,  or  if  there  be  an  excess  of  potas- 
sium compounds  in  any  other  form  than  KG,  the  potassium  combinations 
are  replaced  in  the  organism  by  NaCl,  so  that  new  potassium  and  sodium 
compounds  are  produced  which  are  voided  with  the  urine.  The  organism 
becomes  poorer  in  NaCl,  which  therefore  must  be  taken  in  greater  amounts 
from  the  outside  (Bunge).  This  occurs  habitually  in  herbivora,  and  in 
man  with  vegetable  food  rich  in  potash.  For  human  beings,  and  especially 
for  the  poorer  classes  of  people  who  live  chiefly  on  potatoes  and  foods 
rich  in  potash,  common  salt  is,  under  these  circumstances,  not  only  a  con- 
diment, but  a  necessary  addition  to  the  food  (Bunge  j). 

Lack  of  Alkali  Carbonates  or  Bases  in  the  Food.  The  chemical  processes 
in  the  organism  are  dependent  upon  the  presence  of  tissue-fluids  of  a  cer- 
tain reaction,  and  this  reaction,  which  is  habitually  alkaline  towards  litmus 
and  neutral  towards  phenolphthalein,  is  chiefly  due  to  the  presence  of 
alkali  carbonates  and  carbon  dioxide.  The  alkali  carbonates  are  also  of 
great  importance  not  only  as  a  solvent  for  certain  proteid  bodies  and  as 
constituents  of  certain  secretions,  such  as  the  pancreatic  and  intestinal 
juices,  but  they  are  also  a  means  of  transportation  of  the  carbon  dioxide 
in  the  blood.  It  is  therefore  easy  to  understand  that  a  decrease  below 
a  certain  point  in  the  quantity  of  alkali  carbonate  must  endanger  life.  Such 
a  decrease  not  only  occurs  with  lack  of  bases  in  the  food  which  accelerates 
death  by  a  relatively  great  production  of  acids  through  the  burning  of  the 
proteids,  but  it  also  occurs  when  an  animal  is  given  dilute  mineral  acids  for 
a  period.  In  herbivora  the  fixed  alkalies  of  the  tissues  combine  with  the 
mineral  acids,  and  the  animal  succumbs  in  a  short  time.  In  carnivora 
(and  in  man)  the  bases  of  the  tissues  are  obstinately  retained;  the  mineral 
acids  unite  with  the  ammonia  produced  by  the  decomposition  of  the  pro- 
teids or  their  cleavage  products,  and  carnivora  can  therefore  be  kept  alive 
for  a  longer  time. 

Lack  of  Phosphates  and  Earths.  With  the  exception  of  the  importance 
of  the  alkaline  earths  as  carbonates  and  more  especially  as  phosphates  in 
the  physical  composition  of  certain  structures,  such  as  the  bones  and  teeth, 
their  physiological  importance  is  nearly  unknown.  Nothing  is  known 
positively  in  regard  to  the  need  in  adults  of  phosphates  in  the  food.  In 
experiments  on  rats  with  food  free  from  phosphorus  Gev^erts  2  found  a 
diminution  in  the  phosphorus  excretion  to  TV  of  the  quanity  excreted  dur- 
ing complete  inanition.  The  relationship  of  P:N  changes  also  from  1:10 
to  1 :  100  and  even  still  more.     In  order  to  contain  sufficient  carbon  and 

1  Zeitschr.  f.  Biologie,  9. 

2  Diete  sans  phosphore,  La  Cellule,  18. 


LACK  OF  IRON  IN    THE  FOOD.  037 

nitrogen  with  the  food  man  takes  at  least  ten  times  as  much  phosphorus 
as  is  absolutely  necessary  (Gev.erts).  In  young,  growing  animal-  the 
coin  lit  ions  are  necessarily  different  and  of  special  interest  in  them  is  the 
question  of  the  action  of  an  insufficient  supply  of  earthy  phosphates  and 
alkaline  earths  upon  the  bone  tissue.  This  action  as  well  as  the  various 
results  which  have  been  obtained  by  experiments  on  young  and  old  ani- 
mals, has  already  been  given  in  a  previous  chapter  (X). 

Another  important  question  is,  How  far  do  the  phosphates  take  part  in 
the  construction  of  the  phosphorized  constituents  of  the  body  or  to  what 
extent  are  they  necessary?  The  experiments  of  Rohmaxx  and  his  pupils  ' 
with  phosphorized  (casein,  vitellin)  and  non-phosphorized  proteids  (edes- 
tin)  and  phosphates  show  that  with  the  introduction  of  casein  and  vitellin 
a  deposition  of  nitrogen  and  phosphorus  takes  place,  while  with  non- 
phosphorized  proteid  and  phosphates  this  does  not  seem  to  occur.  The 
body  apparently  does  not  have  the  power  of  building  up  the  phosphorized 
cell  constituents  necessary  for  cell-life  from  non-phosphorized  proteids 
and  phosphates.  On  the  contrary,  according  to  the  observations  of  several 
investigators  the  lecithins  seem  to  possess  this  power. 

Lack  of  Iron.  As  iron  is  an  integral  constituent  of  haemoglobin,  abso- 
lutely necessary  for  the  introduction  of  oxygen,  just  so  is  it  an  indispensable 
constituent  of  food.  Iron  is  a  never-failing  constituent  of  the  nucleins 
and  nucleoproteids,  and  herein  lies  also  another  reason  for  the  necessity 
of  the  introduction  of  iron.  Iron  is  also  of  great  importance  for  the 
action  of  certain  enzymes,  the  oxidases.  In  iron  starvation  iron  is  con- 
tinually eliminated,  even  though  in  diminished  amounts;  and  with  an 
insufficient  supply  of  iron  with  the  food  the  formation  of  haemoglobin 
decreases.  The  formation  of  haemoglobin  is  not  only  enhanced  by  the 
supply  of  organic  iron,  but  also,  according  to  the  general  view,  by 
inorganic  iron  preparations.  The  various  divergent  statements  on  this 
question  have  already  been  given  in  a  previous  chapter  (on  the 
blood). 

In  the  absence  of  protein  bodies  in  the  food  the  organism  must  nourish 
itself  by  its  own  protein  substances,  and  with  such  nutrition  it  must  sooner 
or  later  succumb.  By  the  exclusive  administration  of  fat  and  carbohy- 
drates the  consumption  of  proteids  in  these  cases  is  very  considerably 
reduced.  According  to  the  doctrine  of  C.  Voit,  which  has  been  defended 
by  recent  investigations  of  E.  Voit  and  Korkunoff,2  the  proteid  metab- 
olism is  never  so  slight  under  these  conditions  as  in  starvation.  Accord- 
ing to  several  investigators,  such  as  Hirschfeld,  Kumagawa,  Klem- 
perer,  Sivex,  Landergrex,3  and   others,   the   proteid   metabolism   may 

1  See  Marcuse,  Pfluger's  Arch.,  6",  and  foot-note  1,  page  620. 

2  Zeitschr.  f.  Biologie,  32. 

'Hirschfeld,  Virchow's  Arch.,  114;    Kumagawa,  ibid.,  116;    Klemperer,  Zeitschr. 


638  METABOLISM  WITH   VARIOUS  FOODS. 

indeed,  with  exclusively  fat  and  carbohydrate  diet,  be  smaller  than  in 
complete  starvation.  Thus  Landergren  has  observed  on  an  adult, 
healthy  man  in  nitrogen  starvation  but  with  sufficient  supply  of  energy 
(about  40  calories  per  1  kilo  as  carbohydrates  and  fat)  on  the  fourth  star- 
vation day  that  the  nitrogen  excretion  was  not  more  than  4  grams.  On 
the  seventh  day,  with  only  carbohydrates,  the  nitrogen  excretion  sank  to 
3.34  grams,  which  corresponded  to  0.047  gram  N  per  kilo  of  body  weight 
and  to  0.29  gram  proteid. 

The  absence  of  fats  and  carbohydrates  in  the  food  affect  carnivora  and 
herbivora  somewhat  differently.  It  is  not  known  whether  carnivora  can 
be  kept  alive  for  any  length  of  time  by  food  entirely  free  from  fat  and  carbo- 
hydrates.1 But  it  has  been  positively  demonstrated  that  they  can  be  kept 
alive  a  long  time  by  feeding  exclusively  with  meat  freed  as  much  as  possible 
from  visible  fat  (Pfluger  2) .  Human  beings  and  herbivora,  on  the  con- 
trary, cannot  live  for  any  length  of  time  on  such  food.  On  one  side  they 
lose  the  property  of  digesting  and  assimilating  the  necessarily  large  amounts 
of  meat,  and  on  the  other  a  distaste  for  large  quantities  of  meat  or  proteids 
soon  appears. 

A  question  of  greater  importance  is  whether  it  is  possible  to  maintain 
life  in  an  animal  for  any  length  of  time  with  a  mixture  of  simple  organic 
and  inorganic  foodstuffs.  This  was  not  possible  in  the  experiments  of 
Bunge  and  Lunin,  cited  above.  Recent  investigators,  such  as  Hall  and 
Steinitz,  arrived  at  somewhat  better  results ;  and  Rohmann  3  has  still 
more  recently  arrived  at  still  more  conclusive  results.  He  used  mice  in 
his  experiments,  and  fed  them  with  a  mixture  of  casein,  white  of  egg,  vitel- 
lin,  potato-starch,  wheat-starch,  margarine,  and  salts.  With  this  diet  the 
animals  maintained  their  body  weight  and  brought  forth  young.  These 
latter  could  not  be  raised  on  artificial  food.  A  better  result  was  obtained 
by  adding  some  malt  to  the  food.  It  was  also  possible  to  further  raise 
with  artificial  food  to  maturity,  mice  which  had  been  formed  and  born 
with  artificial  food.  These  mice  remained  somewhat  smaller  than  the  nor- 
mal, and  no  living  young  could  be  obtained  from  them.  If  we  exclude 
the  fact  that  the  foodstuffs  fed  were  not  all  simple  (white  of  egg,  malt),  pure 
foods  it  seems  as  if  artificial  mixtures  of  food  are  sufficient  to  maintain  at 
least  an  adult  animal  for  a  long  time,  while  it  is  not  quite  sufficient  for 
the  development  of  a  young  animal. 


f.  klin.  Med.,  16;  Siven,  Skand.  Arch.  f.  Physiol.,  10  and  11;  Landergren,  1.  c;  foot- 
note 1,  page  630.     See  also  Maly's  Jahresber.,  32. 

1  See  Horbaczewski,  Maly's  Jahresber.,  31,  715. 

z  Pfluger 's  Arch.,  50. 

3  Hall,  Arch.  f.  (Anat.  u.)  Physiol.,  1896;  Steinitz,  Uber  Versuche  mit  kunstlicher 
Ernahrung,  Inaug.-Diss.,  Breslau,  1900;  Rohmann,  Klin,  therap.  Wochenschr. ,  1902, 
No.  40. 


METABOLISM   WITH   VARIOUS  FOODSTUFFS.  639 

IV.   Metabolism  with  Various  Foods. 

For  the  carnivora,  as  above  stated,  meat  as  poor  as  possible  in  fat  may 
be  a  complete  and  sufficient  food.  As  the  proteids  moreover  take  a  special 
place  among  the  organic  nutritive  bodies  by  the  quantity  of  nitrogen  they 
contain,  it  is  proper  that  we  first  describe  the  metabolism  with  an  exclu- 
sively  meat   diet. 

Metabolism  with  food  rich  in  proteids,  or  feeding  only  with  meat  as 
poor  in  fat  as  possible. 

By  an  increased  supply  of  proteids  their  catabolism  and  the  elimina- 
tion of  nitrogen  is  increased,  and  this  in  proportion  to  the  supply  of  proteids. 

If  a  certain  quantity  of  meat  has  been  given  to  carnivora  as  food  daily 
and  the  quantity  is  suddenly  increased,  an  augmented  catabolism  of  pro- 
teids, or  an  increase  in  the  quantity  of  nitrogen  eliminated,  is  the  result. 
If  the  animal  is  fed  daily  for  a  certain  time  with  larger  quantities  of 
the  same  meat,  a  part  of  the  proteids  accumulates  in  the  body,  but  this 
part  decreases  from  day  to  day,  while  there  is  a  corresponding  daily  in- 
crease in  the  elimination  of  nitrogen.  In  this  way  a  nitrogenous  equi- 
librium is  established;  that  is,  the  total  quantity  of  nitrogen  eliminated  is 
equal  to  the  quantity  of  nitrogen  in  the  absorbed  proteids  or  meat.  If, 
on  the  contrary,  an  animal  which  is  in  nitrogenous  equilibrium,  having 
been  fed  on  large  quantities  of  meat,  is  suddenly  given  a  small  quantity 
of  meat  per  day,  then  the  animal  uses  up  its  own  body  proteids,  the 
amount  decreasing  from  day  to  day.  The  elimination  of  nitrogen  and 
the  catabolism  of  proteids  decrease  constantly,  and  the  animal  may  in 
this  case  also  pass  into  nitrogenous  equilibrium,  or  nearly  into  this  con- 
dition.    These  relations  are  illustrated  by  the  following  table  (Voit  *)  : 

Grams  of  Meat  in  the  Food  per  Day. 


1 

Before  the  Test. 
500 

Duri 

ing  the  Test. 
1500 

2 

1500 

1000 

Grams  of  Flesh  Metabolized  in  Body  per  Day. 

1 
1222 
1153 

2                       3                       4                      5 
1310             1390             1410             1440 
1086             1088             1080             1027 

6 
1450 

7 

1500 

In  the  first  case  (1)  the  metabolism  of  meat  before  the  beginning  of  the 
actual  experiment  on  feeding  with  500  grams  of  meat  was  447  grams,  and  it 
increased  considerably  on  the  first  day  of  the  experiment,  after  "feeding  with 
1500  grams  of  meat.  In  the  second  case  (2),  in  which  the  animal  was  pre- 
viously in  nitrogenous  equilibrium  with  1500  grams  of  meat,  the  metab- 
olism of  flesh  on  the  first  day  of  the  experiment,  with  only  1000  grams 
meat,  decreased  considerably,  and  on  the  fifth  day  nearly  a  nitrogenous 

1  Hermann's  Handbuch,  6,  Part  I,  110. 


640  METABOLISM  WITH  VARIOUS  FOODS. 

equilibrium  was  obtained.  During  this  time  the  animal  gave  up  daily- 
some  of  its  own  proteids.  Between  that  point  below  which  the  animal 
loses  from  its  own  weight  and  the  maximum,  which  seems  to  be  depend- 
ent upon  the  digestive  and  assimilative  capacity  of  the  intestinal  canal,  a 
carnivora  may  be  kept  in  nitrogenous  equilibrium  with  varying  quantities 
of  proteids  in  the  food. 

The  supply  of  proteids,  as  well  as  the  proteid  condition  of  the  body, 
affects  the  extent  of  the  proteid  metabolism.  A  body  which  has  become 
rich  in  proteids  by  a  previous  abundant  meat  diet  must,  to  prevent  a  loss 
of  proteids,  take  up  more  proteid  with  the  food  than  a  body  poor  in 
proteids. 

Pettenkofer  and  Voit  have  made  investigations  on  the  metabolism 
of  fat  with  an  exclusively  proteid  diet.  These  investigations  have  shown 
that  by  increasing  the  quantity  of  proteids  in  the  food  the  daily  metab- 
olism of  fat  decreases,  and  they  have  drawn  the  conclusion  from  these 
experiments,  as  detailed  in  Chapter  X,  that  even  a  formation  of  fat 
may  take  place  under  these  circumstances.  The  objections  presented  by 
Pfluger  to  these  experiments,  as  well  as  the  proofs  of  the  formation  of 
fat  from  proteids,  are  also  given  in  the  above-mentioned  chapter. 

According  to  Pfluger 's  doctrine  the  proteid  can  influence  the  forma- 
tion of  fat  only  in  an  indirect  way,  namely,  in  that  it  is  consumed  instead 
of  the  non-nitrogenous  bodies  and  hence  the  fat  and  fat-forming  carbo- 
hydrates are  spared.  If  sufficient  proteid  is  introduced  into  the  food  to 
satisfy  the  total  nutritive  requirements,  then  the  decomposition  of  fat 
stops;  and  if  non-nitrogenous  food  is  taken  at  the  same  time,  this  is  not 
consumed,  but  is  stored  up  in  the  animal  body,  the  fats  as  such,  and  the 
carbohydrates  at  least  in  great  part  as  fat. 

Pfluger  defines  the  "nutritive  requirement"  as  the  smallest  quantity 
of  lean  meat  which  produces  nitrogenous  equilibrium  without  causing  any 
decomposition  of  fat  or  carbohydrates.  At  rest  and  at  an  average  temper- 
ature it  is  found  for  dogs  to  be  2.073  to  2.099  grams  of  nitrogen  1  (in  meat 
fed)  per  kilo  of  flesh  weight  (not  body  weight,  as  the  fat,  which  often 
forms  a  considerable  fraction  of  the  weight  of  the  body,  cannot  as  it  were 
be  used  as  dead  measure) .  Even  when  the  supply  of  proteid  is  in  excess 
of  the  nutritive  requirements,  Pfluger  has  found  that  the  proteid  metab- 
olism increases  with  an  increased  supply  until  the  limit  of  digestive  power 
is  reached,  which  limit  is  about  2600  grams  of  meat  with  a  dog  weighing 
30  kilos.  In  these  experiments  of  Pfluger's  all  of  the  excess  of  proteid 
introduced  was  not  completely  decomposed,  but  a  part  was  retained  by 
the  body.  Pfluger  therefore  defends  the  proposition  "that  a  supply  of 
proteids  only,  without  fat  or  carbohydrate,  does  not  exclude  a  proteid 
fattening. ' ' 

1  See  Schondorff,  Pfluger's  Arch.,  71. 


TISSUE   AND   CIRCULATING  PROTEID.  641 

From  what  has  been  said  on  proteid  metabolism  in  starvation  and  with 
exclusive  proteid  food  it  follows  that  the  proteid  catabolism  in  the  animal 
body  never  stops,  that  the  extent  is  dependent  in  the  first  place  upon  the 
extent  of  proteid  supply,  and  that  the  animal  body  has  the  property,  within 
wide  limits,  of  accommodating  the  proteid  catabolism  to  the  proteid  supply. 

These  and  certain  other  peculiarities  of  proteid  catabolism  have  led 
Voit  to  the  view  that  all  proteids  in  the  body  are  not  decomposed  with 
t  be  same  ease.  Voit  differentiates  the  proteids  fixed  in  the  tissue-elements, 
so-called  organized  proteids,  tissue-protcids,  from  those  proteids  which 
circulate  with  the  fluids  in  the  body  and  its  tissues  and  which  are  taken  up 
by  the  living  cells  of  the  tissues  from  the  interstitial  fluids  washing  them 
and  are  destroyed.  These  circulating  proteids  are,  according  to  Voit,  more 
easily  and  quickly  destroyed  than  the  tissue-proteids.  When,  therefore,  in 
a  fasting  animal  which  has  been  previously  fed  with  meat  an  abundant 
and  quickly  decreasinu-  decomposition  of  proteids  takes  place,  while  in  the 
further  course  of  starvation  this  proteid  catabolism  becomes  less  and 
more  uniform,  this  depends  upon  the  fact  that  the  supply  of  circulating 
proteids  is  destroyed  chiefly  in  the  first  days  of  starvation  and  the  tissue- 
proteids  in  the  last  days. 

The  tissue-elements  constitute  an  apparatus  of  a  relatively  stable  nature, 
which  have  the  power  of  taking  proteids  from  the  fluids  washing  the  tissues 
and  appropriating  them,  while  their  own  proteids,  the  tissue-proteids,  are 
ordinarily  catabolized  to  only  a  small  extent,  about  1  per  cent  daily  (Voit). 
By  an  increased  supply  of  proteids  the  activity  of  the  cells  and  their  ability 
to  decompose  nutritive  proteids  is  also  increased  to  a  certain  degree.  When 
nitrogenous  equilibrium  is  obtained  after  an  increased  supply  of  proteids,  it 
denotes  that  the  decomposing  power  of  the  cells  for  proteids  has  increased 
so  that  the  same  quantity  of  proteids  is  metabolized  as  is  supplied  to  the 
body.  If  the  proteid  metabolism  is  decreased  by  the  simultaneous  admin- 
istration of  other  non-nitrogenous  foods  (see  below),  a  part  of  the  circulat- 
ing proteids  may  have  time  to  become  fixed  and  organized  by  the  tissues, 
and  in  this  way  the  mass  of  the  flesh  of  the  body  increases.  During  starva- 
tion or  with  a  lack  of  proteids  in  the  food  the  reverse  takes  place,  for  a  part 
of  the  tissue  proteids  is  converted  into  circulating  proteids  which  are  metab- 
olized, and  in  this  case  the  flesh  of  the  body  decreases. 

Voit's  theory  has  been  severely  criticised  by  Pfluger.  Pfluger's 
statement,  based  on  an  investigation  made  by  one  of  his  pupils,  Sch«»x- 
dorff,1  is  that  the  extent  of  proteid  destruction  is  not  dependent  upon 
the  quantity  of  circulating  proteids,  but  upon  the  nutritive  condition  of 
the  cells  for  the  time  being — a  view  which  is  not  very  contradictory  of 
Voit  if  the  author  does  not  misunderstand  Pfluger.     Voit  2  has,  as  is 

1  Pfluger,  Pfluger's  Arch.,  54;  Schondorff,  ibid.,  5-4. 
7  Zeitschr.  f.   Biologie,  11. 


642  METABOLISM  WITH  VARIOUS  FOODS. 

known,  stated  that  the  conditions  for  the  destruction  of  substances  in 
the  body  exist  in  the  cells,  and  also  that  the  circulating  proteid,  likewise 
according  to  Voit,  is  first  catabolized  after  having  been  taken  up  by  the 
cells  from  the  fluids  washing  them.  The  point  of  Voit's  theory  is  that 
all  proteids  are  not  destroyed  in  the  body  with  the  same  degree  of  readi- 
ness. The  organized  proteid,  which  is  fixed  by  the  cells  and  has  become 
a  part  of  the  same,  is  destroyed  less  readily,  according  to  Voit,  than  the 
proteid  taken  up  by  the  cells  from  the  nutritive  fluid,  which  serves  as 
material  for  the  chemical  construction  of  the  very  much  more  complicated 
organized  proteids.  This  nutritive  proteid,  which  circulates  with  the 
fluids  before  it  is  taken  up  by  the  cells,  and  which  can  exist  in  store  in 
the  cells  as  well  as  in  the  fluids,  agreeably  to  Voit's  view,  has  been  called 
circulating  proteid  or  supply  proteid  by  him.  It  is  clear  that  these  names 
may  lead  to  misunderstanding,  and  therefore  too  much  stress  should  not 
be  put  upon  them.  The  most  essential  part  of  Voit's  theory  is  the  suppo- 
sition that  the  food  proteid  of  the  cells  is  more  easily  destroyed  than  the 
organized,  real  protoplasmic  proteid,  and  this  assertion  can  hardly,  for 
the  present,  be  considered  as  refuted  or  exactly  proved. 

This  question  is  intimately  connected  with  another,  namely,  whether 
the  food  proteids  taken  up  by  the  cells  are  metabolized  as  such  or  whether 
they  are  first  organized.  The  investigations  of  Pantjm  and  Falck  x  on 
the  transitory  progress  of  the  elimination  of  urea  after  a  meal  rich  in  pro- 
teids throws  light  on  this  question.  From  experiments  upon  a  dog  it  was 
found  that  the  elimination  of  urea  increases  almost  immediately  after  a 
meal  rich  in  proteids,  and  that  it  reaches  its  maximum  in  about  six  hours, 
when  about  one  half  of  the  quantity  of  nitrogen  corresponding  to  the 
administered  proteids  is  eliminated.  If  we  also  recollect  that,  according 
to  an  experiment  of  Schmidt-Mulheim  2  upon  a  dog,  about  37  per  cent  of 
the  given  proteids  are  absorbed  in  the  first  two  hours  after  the  meal  and 
about  59  per  cent  in  the  course  of  the  first  six  hours,  it  may  then  be  inferred 
that  the  increased  elimination  of  nitrogen  after  a  meal  is  due  to  a  catab- 
olization  of  the  digested  and  assimilated  proteids  of  the  food  not  previously 
organized.  If  it  is  admitted  that  the  catabolized  proteid  must  have  been 
organized,  then  the  greatly  increased  elimination  of  nitrogen  after  a 
meal  rich  in  proteids  supposes  a  far  more  rapid  and  comprehensive 
destruction  and  reconstruction  of  the  tissues  than  has  been  generally 
assumed. 

In  this  connection  it  must  be  recalled  that,  according  to  the  very  interest- 

'  Panum,  Nord.  Med.  Arkiv.,  6;  Falck,  see  Hermann's  Handbuch,  6,  Part  I,  107. 
For  further  statements  in  regard  to  the  curve  of  nitrogen  elimination  in  man,  see 
Tschenloff,  Korrespond.  Blatt  Schweiz.  Aerzte,  1896;  Rosemann,  Pfliiger's  Arch.,  65, 
and  Veraguth,  Journ.  of  Physiol.,  21;   Schlosse,  Maly's  Jahresber.,  31. 

2Du  Bois-Reymond's  Arch.,  1879. 


NUTRITIVE   VALVE  OF  GELATINE.  643 

ing  investigations  of  Riazantseff,  substantiated  by  Schepski,  after  par- 
taking  of  food  an  increased  nitrogen  elimination  depends  in  part  upon  the 
increased  work  of  the  digestive  glands.  This  follows  from  the  considerably 
increased  nitrogen  elimination  after  so-called  "apparent  feeding"  (see 
Chapter  l.\  ),  but  has  also  been  confirmed  by  RlAZANTZEFF  '  in  other  ways. 
In  close  connection  with  this  stand  the  observations  of  Xk.wki  and  Zaleski  2 
on  the  free  formation  of  ammonia  in  the  cells  of  the  digestive  apparatus 
during  the  digestion  of  food  rich  in  proteids. 

It  has  been  stated  above  that  other  foods  may  decrease  the  catabolism 
of  proteids.  Gelatine  is  such  a  food.  Gelatine  and  the  gelatine-formers  do 
not  seem  to  be  converted  into  proteid  in  the  body,  and  this  last  cannot 
be  entirely  replaced  by  gelatine  in  the  food.  For  example,  if  a  dog  is  fed 
on  gelatine  and  fat,  its  body  sustains  a  loss  of  proteids  even  when  the 
quantity  of  gelatine  is  so  large  that  the  animal,  with  an  amount  of  fat 
and  meat  containing  just  the  same  quantity  of  nitrogen  as  the  gelatine  in 
question,  may  remain  in  nitrogenous  equilibrium.  On  the  other  hand, 
gelatine,  as  Voir,  Panum  and  Oerum  3  have  shown,  has  a  great  value  as 
a  means  of  sparing  the  proteids,  and  it  may  decrease  the  catabolism  of 
proteids  to  a  still  greater  extent  than  fats  and  carbohydrates.  This  is 
apparent  from  the  following  summary  of  Voit's  experiments  upon  a  dog: 

Food  per  Day.  Flesh. 


Heat.  Gelatine  Fat.  Sugar.  Catabolized.  On  the  Body. 

400  0  200                 0                       450                 -50 

400  0                   0  250                     439                 -39 

400  200                 0                   0                       356                  +44 

I.  Muxk  4  has  lately  arrived  at  similar  results  by  means  of  more  deci- 
sive experiments.  He  found  in  dogs  that  on  a  mixed  diet  which  contained 
3.7  grams  proteid  per  kilo  of  body,  of  which  hardly  3.6  grams  was  catab- 
olized, nearly  &  could  be  replaced  by  gelatine.  The  same  dog  catabolized 
on  the  second  day  of  starvation  three  times  as  much  proteid  as  with  the 
gelatine  feeding.  Muxk  states  also  that  gelatine  has  a  much  greater 
sparing  action  on  proteids  than  the  fats  or  the  carbohydrates. 

This  ability  of  gelatine  to  spare  the  proteids  is  explained  by  Voit  by 
the  fact  that  the  gelatifle  is  decomposed  instead  of  a  part  of  the  circulat- 
ing proteids,  whereby  a  part  of  this  last  may  be  organized. 

The  recent  investigations  of  Krummacher  and  Kirchmaxn  5  show  the 
extent  of  the  sparing  action  of  gelatine  upon  proteids.  The  extent  of 
proteid  destruction  during  gelatine  feeding  was  compared  with  the  extent 


1  Arch,  des  scienc.  biol.  de  St.  Pdtersbourg,  4,  393;  Schepski,  Maly'a  Jahresber.,  30. 

2  See  foot-note  2,  page  34S. 

3  Voit,  1.  c,  123;   Panum  and  Oerum,  Xord.  Med.  Arkiv.,  11. 
*  Pfliiger's  Arch.,  58. 

5  Krummacher,  Zeitschr.  f.  Biologie,  42;    Kirchmann,  ibid.,  40. 


644  METABOLISM  WITH  VARIOUS  FOODS. 

of  proteid  catabolism  in  starvation,  and  it  was  found  that  35-37.5  per 
cent  of  the  quantity  of  proteid  decomposed  in  starvation  could  be  spared 
by  gelatine.  The  physiological  availability  of  gelatine  was  found  by 
Krummacheb  to  be  equal  to  3.88  calories  for  1  gram,  which  corresponds 
to  about  72.4  per  cent  of  the  energy-content  of  the  gelatine. 

Gelatine  may  also  decrease  somewhat  the  consumption  of  fat,  although 
it  is  of  less  value  in  this  respect  than  the  carbohydrates. 

The  question  of  the  nutritive  value  of  proteoses  (and  peptones)  stands 
in  close  relationship  to  the  nutritive  value  of  the  proteids  and  gelatine. 
The  early  investigations  made  by  Malt,  Plos'z  and  Gyergyay,  and 
Adamkiewicz  have  led  to  the  conclusion  that  with  food  which  contains 
no  proteids  besides  peptones  (proteoses)  an  animal  may  not  only  preserve 
its  nitrogenous  equilibrium,  but  its  proteid  condition  may  even  increase. 
According  to  recent  and  more  exact  investigations  by  Pollitzer,  Zuntz, 
and  Muxk  the  proteoses  have  the  same  nutritive  value  as  proteids,  at 
least  in  short  experiments.  According  to  Pollitzer  this  is  true  for  differ- 
ent proteoses  as  well  as  for  true  peptone;  but  this  does  not  correspond 
with  the  experience  of  Ellinger,1  who  finds  that  the  true  antipeptone 
(gland  peptone)  is  not  able  to  entirely  replace  proteids  or  to  prevent  the 
loss  of  proteid  in  the  animal  body.  On  the  contrary,  according  to  him,  it 
has,  like  gelatine,  the  property  of  sparing  proteids.  Voit  long  ago  ex- 
pressed a  similar  view.  According  to  him  the  proteoses  and  peptone  may 
indeed  replace  the  proteids  for  a  short  time,  but  not  permanently;  they 
can  spare  the  proteids,  but  cannot  be  converted  into  proteids.  According 
to  the  researches  of  Blum  2  the  different  proteoses  have  various  nutritive 
values.  In  his  experiments  the  heteroproteose  from  fibrin  could  not 
replace  the  proteids  of  the  food,  while  casein  protoproteose  had  this 
property. 

The  question  as  to  the  nutritive  value  of  proteoses  and  peptones  has 
turned  in  a  new  direction,  due  to  the  more  recent  views,  as  mentioned  in 
Chapter  IX,  on  the  absorption  of  proteids  where  the  proteids  are  not  ab- 
sorbed chiefly  as  proteoses  and  peptones,  but  as  simpler  cleavage  products. 
Loewi3  has  attempted  by  special  experiments  to  bring  about  a  proteid 
synthesis  from  these  simple  products  in  the  body.  Even  if  this  view 
is  correct  and  if  the  greatest  portion  of  the  food-proteid  is  split,  before 
absorption,  into  simpler  products  than  peptone,  it  does  not  follow  that  the 


1  Maly,  Pfliiger's  Arch.,  9;  Plos'z  and  Gyergyay,  ibid., 10;  Adamkiewicz,  "Die 
Xatur  und  der  Xiihrwerth  des  Peptons"  (Berlin,  1877);  Pollitzer,  Pfliiger's  Arch., 
37,  301;  Zuntz,  ibid.,  37,  313;  Munk,  Centralbl.  f.  d.  med.  Wissensch.,  1889,  20,  and 
Deutsch.  med.  Wochenschr.,  1889;    Ellinger,  Zeitschr.  f.  Biologie,  33  (literature). 

2Zeitschr.  f.  physiol.  Chem.,  30;   Voit,  1.  c,  394. 

3  Arch.  f.  exp.  Path.  u.  Pharm.,  48.  See  also  Henderson  and  Dean,  Amer.  Journ. 
of  Physiol.,  9,  and  Plumier,  Chem.  Centralbl.,  1903,  1,  410. 


SPARING   ACTION  OF  FATS  AND  CARBOHYDRATES.  845 

proteoses  and  peptones  also  can  completely  replace  the  proteids  of  the 
food.  The  proteoses  and  peptones  are  formed  by  cleavages,  and  perhaps 
certain  atomic  complexes  are  absent  which  occur  in  the  mixture  of  cleav- 
age products  and  which  are  necassary  for  a  regeneration  of  special  proteid 
bodies. 

From  experiments  made  by  Weiske  and  others  on  herbivora  it  appears 
that  asparagin  may  spare  proteid  in  such  animals.  According  to  Kbll- 
KEB  '  the  sparing  action  of  asparagin  is  only  of  an  indirect  kind  because 
it  serves  as  nutrition  for  the  bacteria  in  the  digestive  tract  instead  of  the 
proteids,  and  also  the  recent  investigations  of  Kellxer  and  collaborators 
show  that  asparagin  can  only  spare  the  proteid  catabolism  in  ruminants 
with  food  poor  in  proteid  but  rich  in  carbohydrates.  In  carnivora  (I. 
Mink)  and  in  mice  (Voit  and  Politis)  it  was  found  that  asparagin  has 
only  a  very  slight,  if  any,  sparing  action  on  the  proteids.  It  is  not  known 
how  it  acta  in  man. 

Metabolism  on  a  Diet  consisting  of  Proteid,  with  Fat  or  Carbohydrates. 
Fat  cannot  arrest  or  prevent  the  catabolism  of  proteids;  but  it  can  decrease 
it,  and  so  spare  the  proteids.  This  is  apparent  from  the  following  table  of 
Voit.2    A  is  the  average  for  three  days,  and  B  for  six  days. 

Food.  Flesh. 


Meat.  Fat.  Metabolized.       On  the  Body. 

A 1500  0  1512  -12 

B 1500  150  1474  +26 

According  to  Voit  the  adipose  tissue  of  the  body  acts  like  the  food-fat, 
and  the  proteid-sparing  effect  of  the  former  may  be  added  to  that  of  the 
latter,  so  that  a  body  rich  in  fat  may  not  only  remain  in  nitrogenous  equi- 
librium, but  may  even  add  to  the  store  of  body  proteids,  while  in  a  lean 
body  with  the  same  food  containing  the  same  amount  of  proteids  and  fat 
there  would  be  a  loss  of  proteids.  In  a  body  rich  in  fat  a  greater  quantity 
of  proteids  is  protected  from  metabolism  by  a  certain  quantity  of  fat  than 
in  a  lean  body. 

Because  of  the  sparing  action  of  fats  an  animal  to  whose  food  fat  is 
added  may.  as  is  apparent  from  the  table,  increase  its  store  of  proteid 
with  a  quantity  of  meat  which  is  insufficient  to  preserve  nitrogenous  equi- 
librium. 

Like  the  fats  the  carbohydrates  have  a  sparing  action  on  the  proteids. 
By  the  addition  of  carbohvdrates  to  the  food  the  carnivora  not  onlv  remains 


1  Weiske,  Zeitschr.  f.  Biologie,  15  and  17,  and  Centralbl.  f.  d.  med.  Wissensch., 
1890,  045;  Munk,  Virchow's  Arch.,  94  and  98;  Politis,  Zeitschr.  f.  Biologie,  28.  See 
also  Mauthner,  ibid.,  28;  Gabriel,  ibid.,  29;  and  Voit,  ibid.,  29,  125;  Kellner,  Maly's 
Jahresber.,  27,  and  Zeitschr.  f.  Biologie,  39. 

2  Voit  in  Hermann 's  Handbuch,  6,  130. 


646  METABOLISM  WITH  VARIOUS  FOODS. 

in  nitrogenous  equilibrium,  but  the  same  quantity  of  meat  which  in  itself 
is  insufficient  and  which  without  carbohydrates  would  cause  a  loss  of  weight 
in  the  body  may  with  the  addition  of  carbohydrates  produce  a  deposit  of 
proteids.     This  is  apparent  from  the  following  table: 1 

Food.  Flesh. 


Meat. 

Fat. 

Sugar. 

Starch. 

Metabolized 

On  the  Bodj 

500 

250 

558 

-    58 

500 

300 

466 

+   34 

500 

200 

505 

-     5 

800 

250 

745 

+  55 

800 

200 

773 

+   27 

2000 

200^300 

1792 

+  208 

2000 

250 

1883 

+  117 

The  sparing  of  proteid  by  carbohydrate  is  greater,  as  shown  by  the 
table,  than  by  fats.  According  to  Voit  the  first  is  on  an  average  9  per 
cent  and  the  other  7  per  cent  of  the  administered  proteid  without  a  previ- 
ous addition  of  non-nitrogenous  bodies.  Increasing  quantities  of  carbo- 
hydrates in  the  food  decrease  the  proteid  metabolism  more  regularly  and 
constantly  than  increasing  quantities  of  fat. 

Because  of  this  great  proteid-sparing  action  of  carbohydrates  the  her- 
bivora,  which  as  a  rule  partake  of  considerable  quantities  of  carbohydrates, 
assimilate  proteids  readily  (Voit). 

The  greater  proteid-sparing  action  of  carbohydrates  as  compared  to 
that  of  the  fats  occurs,  as  shown  by  Landergren,2  to  a  still  higher  degree 
with  food  poor  in  nitrogen  or  in  nitrogen  starvation,  in  which  cases  the 
carbohydrates  have  double  the  proteid-sparing  action  as  compared  to  an 
isodynamic  quantity  of  fat. 

The  proteid-sparing  action  of  the  carbohydrates  and  fats  has  generally 
been  studied  by  the  one-sided  feeding  with  one  or  the  other  of  these  two 
groups  of  foodstuffs.  The  question  may  be  raised  whether  the  difference 
observed  between  the  fats  and  carbohydrates  could  not  be  brought  about 
also  by  the  simultaneous  supply  of  carbohydrates  and  fat  in  varying  pro- 
portions. Tallquist  3  has  made  a  series  of  experiments  on  this  subject. 
In  one  of  the  periods  16.27  grams  N,  44  grams  fat,  and  466  grams  carbo- 
hydrate were  given;  in  a  second  16.08  grams  N,  140  grams  fat,  and  250 
grams  carbohydrate,  containing  nearly  the  same  number  of  calories,  namely, 
2S67  and  2873  calories.  In  both  cases  nearly  a  complete  nitrogenous 
equilibrium  was  reached  and  the  carbohydrate  did  not  spare  more  proteid 
than  the  fat.  It  is  therefore  possible  that  the  fat  has  about  the  same 
proteid-sparing  action  as  an  isodynamic  amount  of  carbohydrate  when  the 
quantity  of  carbohydrates  does  not  sink  below  a  certain  minimum,  which 
is  not  known  for  the  present. 

1  Voit  in  Hermann's  Handbuch,  6,  143. 

2L.  c,  Inaug.-Diss.,  and  Skand.  Arch.  f.  Physiol.,  14. 

8  Finska  Lakaresiillskapets  handl.,  1901.     See  also  Arch.  f.  Hygiene,  41. 


LIMIT  OF  PROTEID  CATABOLISM.  017 

This  condition  as  well  as  the  extent  of  proteid-sparing  action  of  the 
carbohydrates  stands,  according  to  Landergben,1  in  close  relation  to  the 
formation  of  sugar  in  the  body.    The  animal  body  always  needs  sugar, 

and  a  lack  of  carbohydrates  in  the  food  leads  to  a  part  of  the  proteids  being 
used  in  the  sugar  formation.  This  part  can  be  spared  by  carbohydrates 
but  not  by  fats,  from  which,  according  to  Landergren,  the  carbohydrates 
cannot  be  formed.  In  tills  lies  also  the  probable  reason  why  the  fats,  on 
being  led  exclusively  but  not  with  a  sufficient  supply  of  carbohydrates, 
have  a  much  lower  proteid-sparing  action  than  the  carbohydrates.  The 
fats  cannot  prevent  the  proteid  catabolism  necessary  for  the  formation  of 
sugar  on  a  diet  lacking  carbohydrates. 

The  law  as  to  the  increased  proteid  catabolism  with  increased  proteid 
supply  applies  also  to  food  consisting  of  proteid  with  fat  and  carbohydrates. 
In  these  cases  the  body  tries  to  adapt  its  proteid  catabolism  to  the  supply; 
and  when  the  daily  calorie-supply  is  completely  covered  by  the  food,  the 
organism  can,  within  wide  limits,  be  in  nitrogenous  equilibrium  with  dif- 
ferent quantities  of  proteid. 

The  upper  limit  to  the  possible  proteid  catabolism  per  kilo  and  per  day 
has  only  been  determined  for  herbivora.  For  human  beings  it  is  not 
known,  and  its  determination  is  from  a  practical  standpoint  of  secondary 
importance.  What  is  more  important  is  to  ascertain  the  lower  limit,  and 
on  this  subject  wo  have  several  experiments  upon  man  as  well  as  upon 
dogs  by  Hirschfeld,  Kumagawa,  Klemperer,  Munk,  Rosenheim,2  and 
others.  It  follows  from  these  experiments  that  the  lower  limit  of  proteid 
needed  for  human  beings  for  a  week  or  less  is  about  30-40  grams  or  0.4- 
0.6  gram  per  kilo  with  a  body  of  average  weight,  v.  Xoorden  3  considers 
0.6  gram  proteid  (absorbed  proteid)  per  kilo  and  per  day  as  the  lower 
limit.  The  above-mentioned  figures  are  only  valid  for  short  series  of  ex- 
periments ;  still  there  exist  the  observations  of  E.  Voit  and  Constantixidi  * 
on  the  diet  of  a  vegetarian  in  which  the  proteid  condition  was  kept  nearly 
but  not  completely  for  a  long  time  with  about  0.6  gram  of  proteid  per  kilo. 

According  to  Yoit's  normal  figures,  which  will  be  spoken  of  below,  for 
the  nutritive  need  of  man,  an  average  working  man  of  about  70  kilos 
weight  requires  on  a  mixed  diet  about  40  calories  per  kilo  (true  calories 
or  net  calories,  i.e.,  the  combustion  value  of  the  absorbed  foods).  In 
the  above  experiments  with  food  very  poor  in  proteid  the  demand  for 
calories  was  considerably  greater;  as,  for  instance,  in  certain  cases  it  was 
51  (Kumagawa)  or  even  7S.5  calorics  (Klemperer).      It  therefore  seems 

1  L.  c,  Inaug.-Diss.     See  also  Skand.  Arch.  f.  Physiol.,  14. 

3  See  foot-note  3,  page  637;  also  Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1S91  and  1S96$ 
Rosenheim,  ibid.,  1891;  Pfliiger's  Arch.,  54. 

3  Grundriss  einer  Methodik  der  Stoffwechseluntersuchungen.     Berlin,  1892. 

4  Zeitschr.  f.  Biologie,  25. 


648  METABOLISM  WITH  VARIOUS  FOODS. 

as  if  the  above  very  low  supply  of  proteid  was  only  possible  with  great 
waste  of  non-nitrogenous  food;  but  in  opposition  to  this  it  must  be. 
recalled  that  in  Voit  and  Const antinidi's  experiments  upon  the  vege- 
tarian, who  for  years  was  accustomed  to  a  food  very  poor  in  proteid  and. 
rich  in  carbohydrate,  the  calories  amounted  to  only  43.7  per  kilo. 

Recently  Siven  has  shown  by  experiments  upon  himself  that  the 
adult  human  organism,  at  least  for  a  short  time,  can  be  maintained  in  nitrog- 
enous equilibrium  with  a  specially  low  supply  of  nitrogen  without  increas- 
ing the  calories  in  the  food  above  the  normal.  With  a  supply  of  41-43 
calories  per  kilo  he  remained  in  nitrogenous  equilibrium  for  four  days 
with  a  supply  of  nitrogen  of  0.08  gram  per  kilo  of  body  weight.  Of  the 
nitrogen  taken,  a  part  was  of  a  non-pro teid  nature  and  the  quantity  of 
true  proteid  nitrogen  was  only  0.045  gram,  corresponding  to  about  0.3  gram 
of  proteid  per  kilo  of  body  weight.  That  this  low  limit,  which  by  the  way 
only  holds  for  a  short  time,  has  no  general  validity  follows  from  other  observa- 
tions. Thus  Caspari  1  also,  in  an  experiment  on  himself,  could  not  attain 
complete  nitrogenous  equilibrium  on  a  much  greater  nitrogen  supply. 
The  proteid  minimum  seems  also  to  be  different  for  various  individuals. 

The  very  important  question  as  to  the  conditions  favoring  the  depo- 
sition of  fat  and  flesh  in  the  body  is  closely  associated  with  what  has 
just  been  said  in  regard  to  foods  consisting  of  proteid  and  non-nitrogenous 
foodstuffs.  In  this  connection  it  must  be  remembered  in  the  first  place 
that  all  fattening  presupposes  an  overfeeding,  i.e.,  a  supply  of  foodstuffs 
which  is  greater  than  that  catabolized  in  the  same  time. 

In  carnivora  a  flesh  deposition  may  take  place  on  the  exclusive  feeding 
with  meat.  This  is  not  generally  large  in  proportion  to  the  quantity  of 
proteid  catabolized.  As  shown  by  an  experiment  upon  a  male  cat  by 
Pfluger  2  this  may  be  so  great  that  the  body  doubles  in  weight  under 
favorable  conditions.  In  man  and  herbivora,  on  the  contrary,  the  demand 
for  calories  may  not  be  covered  by  proteid  alone,  and  the  question  as  to 
the  conditions  of  fattening  with  a  mixed  diet  is  of  importance. 

These  conditions  have  also  been  studied  in  carnivora,  and  here,  as 
Voit  has  shown,  the  relationship  between  proteid  and  fat  (and  carbo- 
hydrates) is  of  great  importance.  If  much  fat  is  given  in  proportion  to. 
the  proteid  of  the  food,  as  with  average  quantities  of  meat  with  consider- 
able addition  of  fat,  then  nitrogenous  equilibrium  is  only  slowly  attained 
and  the  daily  deposit  of  flesh,  though  not  large,  is  quite  constant,  and 
may  become  greater  in  the  course  of  time.  If,  on  the  contrary,  much  meat 
besides  proportionately  little  fat  is  given,  then  the  deposit  of  proteid  with 
increased  catabolism  is  smaller  day  by  da)7,  and  nitrogenous  equilibrium 

1  Siv£n,  Skand.  Arch.  f.  Physiol.,  10  and  11;  Caspari,  Arch.  f.  (Anat.  u.)  Pbysiol., 
1901. 

2  Pfluger's  Arch.,  77. 


DEPOSITION  OF  FLESH. 


649 


is  attained  in  a  few  days.  In  spite  of  the  somewhat  larger  deposit  per 
diem,  the  total  flesh  deposit  is  not  considerable  in  these  cases.  The  fol- 
lowing experiment  of  VoiT  may  serve  as  example: 


Number  of 

Days  of  Kx- 

periinent;it  ion. 

Food. 

Total 

Deposit  of 

Flesh. 

Daily 

Deposit  of 

Flesh. 

Nitrogenous 

Meat,  Grams. 

Fat,  Grams. 

Equilibrium. 

32 

7 

1800 

250 
250 

1792 
854 

56 
122 

not  attained 
attained 

The  greatest  absolute  deposition  of  flesh  in  the  body  was  obtained  in 
these  cases  with  only  500  grams  of  meat  and  250  grams  of  fat,  and  even  after 
32  days  nitrogenous  equilibrium  had  not  occurred.  On  feeding  with  1S00 
grains  of  meat  and  250  grams  of  fat  nitrogenous  equilibrium  was  established 
after  seven  days;  and  though  the  deposition  of  flesh  per  day  was  greater, 
still  the  absolute  deposit  was  not  one  half  as  great  as  in  the  former  case. 

The  experiments  of  Krug  upon  himself,  under  the  direction  of  v.  Noor- 
den,  give  us  information  as  to  the  practicability  of  flesh  deposition  in  man.  • 
With  abundant  food  (2590  cal.  =  44  cal.  per  kilo)  Krug  was  close  to  nitrog- 
enous equilibrium  for  six  days.  He  then  increased  the  nutritive  supply 
to  4300  cal.  =  71  cal.  per  kilo  for  fifteen  days  by  the  addition  of  fat  and 
carbohydrate,  and  in  this  time  309  grams  of  proteid,  corresponding  to  1455 
grams  of  muscle,  was  spared.  Of  the  excess  of  administered  calories  in  this 
case  only  5  per  cent  was  used  for  flesh  deposit  and  95  per  cent  for  fat 
deposit.  On  the  other  hand  Bornstein,1  also  experimenting  upon  him- 
self, could  produce,  without  any  considerable  increase  in  calories,  an  increase 
in  the  proteid  condition  of  about  100  grams  of  proteid,  corresponding  to  500 
grams  of  flesh,  in  the  course  of  fourteen  days  simply  by  increasing  the  supply 
of  proteid  (50  grams  of  nutrose  =  sodium  casein  with  7  grams  N  per  day). 
We  cannot  state  whether  such  a  nitrogen  or  proteid  retention  indicates  a 
true  flesh  deposition,  i.e.,  the  re-formation  of  living  tissue  or  not. 

Bornstkix  arrived  at  still  better  results  in  regard  to  proteid  retention 
by  simultaneous  muscle  work,  as  in  these  cases  the  nitrogen  retention 
corresponded  to  a  flesh  deposit  of  800  grams.  The  importance  of  work 
for  the  so-called  proteid  deposition  follows  also  from  many  other  obser- 
vations, and  it  is  in  agreement  with  daily  experience  that  a  man  cannot 
be  made  muscle-strong  by  superfeeding  alone.  A  work-hypertrophy 
must  also  be  introduced. 

It  is  difficult  to  produce  a  permanent  flesh  deposit  in  man  by  overfeed- 
ing alone.     Flesh  deposition  is,  according  to  v.  Noorden,  a  function  of 

1  Krug,  cited  from  v.  Noorden,  Lehrbuch  der  Patholopie  des  Stoffwechsel.,  120; 
Bornstein,  Berl.  klin.  Wochenschr.,  1S98,  and  Pfiiiger's  Arch.,  88. 


650  METABOLISM  WITH  VARIOUS  FOODS. 

the  specific  energy  of  the  developing  cells  and  the  cell-work  to  a  much  higher 
extent  than  the  excess  of  food.  Therefore  there  is  observed,  according  to 
v.  Noorden,  abundant  flesh  deposition  (1)  in  each  growing  body;  (2)  in 
those  no  longer  growing  but  whose  body  is  accustomed  to  increased  work; 
(3)  whenever,  by  previous  insufficient  food  or  by  disease,  the  flesh  condi- 
tion of  the  body  has  been  diminished  and  therefore  requires  abundant 
food  to  replace  the  same.  The  deposition  of  flesh  is  in  this  case  an 
expression  of  the  regenerative  energy  of  the  cells. 

The  experiences  of  graziers  show  that  in  food-animals  a  flesh  deposit 
does  not  occur,  or  at  least  is  only  inconsiderable,  on  overfeeding.  The 
individuality  and  the  race  of  the  animal  are  of  importance  for  flesh  depo- 
sition.1 

As  above  stated  (Chapter  X)  respecting  the  formation  of  fat  in  the  animal 
body,  the  most  essential  condition  for  a  fat  deposition  is  an  overfeeding 
with  non-nitrogenous  foods.  The  extent  of  fat  deposition  is  determined  by 
the  excess  of  calories  administered  over  those  actually  needed.  If  a  large 
part  of  the  calorie-demand  is  covered  by  proteid,  then  a  greater  part  of 
the  non-nitrogenous  foodstuffs  simultaneously  ingested  is  spared,  i.e.,  used 
for  fat  deposition.  But  as  proteid  and  fat  are  expensive  nutritive  bodies 
as  compared  with  carbohydrates,  the  supply  of  greater  quantities  of  carbo- 
hydrates is  important  for  fat  deposition.  The  body  decomposes  less  sub- 
stance at  rest  than  during  activity.  Bodily  rest,  besides  a  proper  com- 
bination of  the  three  chief  groups  of  organic  foods,  is  therefore  also  an 
essential  requisite  for  an  abundant  fat  deposit. 

Action  of  Certain  Other  Bodies  on  Metabolism.  Water.  If  a  quantity 
in  excess  of  that  which  is  necessary  is  introduced  into  the  organism,  the 
excess  is  quickly  and  principally  eliminated  with  the  urine.  This  in- 
creased elimination  of  urine  causes  in  fasting  animals  (Voit,  Forster), 
but  not  to  any  appreciable  degree  in  animals  taking  food  (Seegen,  Sal- 
kowski  and  Munk,  Mayer,  Dubelir2),  an  increased  elimination  of  urea. 
The  reason  for  this  increased  excretion  is  to  be  found  in  the  fact  that  the 
drinking  of  much  water  causes  a  complete  washing  out  of  the  urea  from  the 
tissues.  Another  view,  which  is  defended  by  Voit,  is  that  because  of  the 
more  active  current  of  fluids,  after  taking  large  quantities  of  water  an  in- 
creased metabolism  of  proteids  takes  place.  Voit  considers  this  explana- 
tion the  correct  one,  although  he  does  not  deny  that  by  the  liberal  admin- 
istration of  water  a  more  complete  washing  out  of  the  urea  from  the  tissues 
takes   place.     More  recent  investigations   of  Neumann  3  show  that  the 

1  See  also  Svenson,  Zeitschr.  f.  klin.  Med.,  43. 

2  Voit,  Untersucb.  iiber  den  Einfluss  des  Kochsalzes,  etc.  (Miinchen,  1860) ;  Forster, 
cited  from  Voit  in  Hermann's  Handbuch,  6,  153;  Seegen,  Wien.  Sitzungsber.,  63; 
Salkowski  and  Munk,  Virchow's  Arch.,  71;  Mayer,  Zeitschr.  f.  klin.  Med.,  2;  Dubelir, 
Zeitschr.  f.  Biologic,  28. 

3  Arch.  f.  Hygiene,  36. 


ACTION  OF   WATER,  SALTS,  ETC.,   UPON  METABOLISM.       661 

increased  nitrogen  excretion  is  in  fact  due  to  an  increased  lixiviation  of 
the  tissues. 

When  the  body  has  lost  a  certain  amount  of  water,  then  the  abstinence 
from  water  (in  animals)  is  accompanied  by  a  rise  in  the  proteid  metabo- 
lism (Landauer,  Straub  l).  In  regard  to  the  action  of  water  on  the  for- 
mation of  fat  and  its  metabolism,  the  view  that  the  free  drinking  of  water 
is  favorable  for  the  deposition  of  fat  seems  to  be  generally  admitted,  while 
the  drinking  of  only  very  little  water  acts  against  its  formation. 

Salts.  The  statements  are  somewhat  contradictory  in  regard  to  the 
action  of  salts,  for  example  sodium  chloride  and  the  neutral  salts,  which 
partly  depends  upon  the  use  of  large  and  varying  amounts  of  salt  in  the  ex- 
periments. Recent  investigations  of  Straub  and  Rost  2  have  shown  that 
the  action  of  salts  stands  in  close  relationship  to  their  power  of  abstracting 
water.  Small  amounts  of  salt  which  do  not  produce  diuresis  have  no  action 
on  metabolism.  On  the  contrary,  larger  amounts  which  bring  about  a 
diuresis  which  is  not  compensated  by  the  ingestion  of  water,  produce  a  rise 
in  the  proteid  metabolism.  If  the  diuresis  is  compensated  by  drinking 
water,  then  the  proteid  metabolism  is  not  increased  by  salts,  but  is 
diminished  to  a  slight  degree.  An  increased  nitrogen  excretion  caused 
by  taking  salts  can  be  somewhat  increased  by  the  ingestion  of  water  and 
thus  increasing  the  diuresis,  and  the  action  of  salts  seems  to  bear  a  close 
relationship  to  the  demand  and  supply  of  water. 

Alcohol.  The  question  as  to  how  far  the  alcohol  absorbed  in  the  intes- 
tinal canal  is  burnt  in  the  body,  or  whether  it  leaves  the  body  unchanged 
by  various  channels,  has  been  the  subject  of  much  discussion.  To  all 
appearances  the  greatest  part  of  the  alcohol  introduced  (95  per  cent)  is 
burnt  in  the  body  (Subbotin,  Thudichum,  Bodlander,  Benedicenti  3). 
As  the  alcohol  has  a  high  calorific  value  (1  gram  =7  calories),  then  the  ques- 
tion arises  whether  it  acts  sparingly  on  other  bodies,  and  whether  it  is  to 
be  considered  as  a  nutritive  substance.  The  older  investigations  made  to 
decide  this  question  have  led  to  no  decisive  result.  The  thorough  investi- 
gations of  Atwater  and  Benedict,  Zuxtz  and  Geppert,  Bjerre,  Clo 
patt,  Neumann,  Offer,  Rosemann/  and  others,  seem  to  show  positively 
that  in  man  alcohol  can  diminish  the  consumption  not  only  of  fat  and 
carbohydrates,  but  also  the  proteids,  although  at  first,  due  to  its  poisonous 
properties,  it  may  increase  the  proteid  metabolism  for  a  short  time.  The 
nutritive  value  of  alcohol  can  only  be  of  special  importance  in  certain 
cases,  as  large  amounts  of  alcohol  taken  at  one  time,  or  the  continued  use 

1  Landauer.  Maly's  Jahresber.,  24;   Straub,  Zeitschr.  f.  Biologic,  37. 
1 W.  Straub,  Zeitschr.  f.   Biologic,  3"  and  38;    Rost,   Arbeiten  aua  d.   Kaiserliche 
Gesundheitsamte,  18  (literature).     See  also  Gruber,  Maly's  Jahresber.,  30,  012. 

3  Arch.  f.  (Anat.  a.)  Physiol.,  1896,  which  contains  the  literature. 

4  In  regard  to  the  literature  on  this  subject,  see  the  works  of  0.  Neumann,  Arch.  f. 
Hygiene,  36  and  11,  and  Rosemann,  Pfhiger's  Arch.,  SO  and  91. 


052  METABOLISM  WITH  VARIOUS  FOODS. 

of  smaller  quantities,  has  an  injurious  action  on  the  organism.  Alcohol 
may  therefore  be  regarded  as  a  foodstuff  only  in  exceptional  cases,  and  in 
other  respects  must  be  considered  as  an  article  of  luxury. 

Coffee  and  tea  have  no  action  on  the  exchange  of  material  which  can  be 
positively  proved,  and  their  importance  lies  chiefly  in  their  action  upon  the 
nervous  system.  It  is  impossible  to  enter  into  the  effect  of  various  thera- 
peutic agents  upon  metabolism. 

V.  The  Dependence  of  Metabolism  on  Other  Conditions. 

The  so-called  abstinence  value  which  was  previously  mentioned,  i.e., 
the  extent  of  metabolism  with  absolute  rest  of  body  and  inactivity  of  the 
intestinal  tract,  serves  best  as  a  starting-point  for  the  study  of  metabolism 
under  various  external  circumstances.  The  metabolism  going  on  under 
these  conditions  leads  in  the  first  place  to  the  production  of  heat,  and  it  is 
only  to  a  subordinate  degree  dependent  upon  the  work  of  the  circulatory 
and  respiratory  apparatus  and  the  activity  of  the  glands.  According  to  a 
calculation  by  Zuntz,1  only  10-20  per  cent  of  the  total  calories  of  the 
abstinence  value  belongs  to  the  circulation  and  respiration  work. 

The  magnitude  of  the  abstinence  value  depends  in  the  first  place  upon 
the  heat  production  necessary  to  cover  the  loss  of  heat,  and  this  heat  pro- 
duction is  in  turn  dependent  upon  the  relationship  between  the  weight 
and  the  surface  of  the  body. 

Weight  of  Body  and  Age.  The  greater  the  mass  of  the  body  the  greater 
the  absolute  consumption  of  material;  while,  on  the  contrary,  other  things 
being  equal,  a  small  individual  of  the  same  species  of  animal  metabolizes 
absolutely  less,  but  relatively  more  as  compared  with  the  unit  of  the  weight 
of  the  body.  It  must  be  remarked  that  the  relation  between  flesh  and  fat 
in  the  body  exerts  an  important  influence.  The  extent  of  the  metabolism 
is  dependent  upon  the  quantity  of  active  cells,  and  a  very  fat  individual 
therefore  decomposes  less  substance  per  kilo  than  a  lean  person  of  the 
same  weight.  According  to  Rubner  2  the  importance  of  the  size  of  the 
flesh-  or  cell-mass  in  the  body  is  overestimated.  In  his  investigations  on 
two  boys,  one  of  whom  was  corpulent  and  the  other  normally  developed, 
and  on  comparing  the  food-need  with  that  found  by  Camerer  for  boys 
of  the  same  weight,  Rubner  came  to  the  result  that  the  exchange  of  force 
in  the  corpulent  boy  almost  completely  corresponded  with  that  in  the  non- 
corpulent  boy  of  the  same  weight.  By  approximately  estimating  the  quan- 
tity of  fat  in  the  body  Rubner  was  also  able,  from  the  proteid  condition,  to 
compare  the  calculated  exchange  of  energy  with  that  actually  found.  The 
exchange  per  kilo  amounted  to  52  calories  in  the  lean  and  43.6  cal.  in  the 

1  Cited  from  v.  Noorden's  Handhuch,  97. 

2  Beitriige  zur  Ernahrung  im  Knabenalter,  etc.    Berlin,  1902. 


INFLUENCE  OF  SURFACE   UPON  METABOLISM.  653 

fat  boy,  while,  if  the  proteid  condition  was  a  measure,  one  would  expect  an 
exchange  of  calories  of  only  35  cal.  for  the  fat  person.  We  cannot  there- 
fore admit  of  a  diminished  activity  of  the  cell-mass  in  the  fat  boy,  but 
rattier  an  increased  activity.  According  to  Rubner  it  Is  not  the  flesh- 
mass  (proteid  mass)  alone,  but  its  variable  functional  changes,  which  deter- 
mine the  extent  of  decomposition.  In  women,  who  generally  have  Lesa 
body  weight  and  a  greater  quantity  of  fat  than  men,  the  metabolism  in 
general  is  smaller,  and  the  latter  is  ordinarily  about  four-fifths  that  of 
men. 

The  question  as  to  what  extent  gender  specially  influences  metabolism 
remains  to  be  investigated.  Tigerstedt  and  Sonden  1  found  that  in  the 
young  the  carbon-dioxide  elimination,  per  kilo  of  body  weight  as  well  as 
per  square  meter  of  body  surface,  was  considerably  greater  in  males  than 
in  females  of  the  same  age  and  the  same  weight  of  body.  This  difference 
between  the  two  sexes  seems  to  disappear  gradually,  and  at  old  age  it 
is  entirely  absent.2 

The  essential  reason  why  small  animals  decompose  relatively  more  sub- 
stance than  large  ones,  when  calculated  per  kilo  body  weight,  is  that  the 
bodies  of  smaller  animals  have  greater  surface  in  proportion  to  their  mass. 
On  this  account  the  loss  of  heat  is  greater,  which  causes  increased  heat  pro- 
duction, i.e.,  a  more  active  metabolism.  This  is  also  the  reason  why  young 
individuals  of  the  same  kind  show  a  relatively  greater  decomposition  than 
older  ones.  If  the  heat  production  and  carbon-dioxide  elimination  is  cal- 
culated on  the  unit  of  surface  of  the  body,  we  find,  on  the  contrary,  as  the 
experiments  of  Rubner,  Richet,3  and  others  show,  that  they  vary  only 
slightly  from  a  certain  average  in  individuals  of  different  weights. 

According  to  Rubner  's  ride  as  to  the  influence  of  the  surface,  which 
has  been  recently  formulated  by  E.  Voit,  the  need  of  energy  in  homoio- 
thermic  animals  is  influenced  by  the  development  of  their  surface  when 
their  body  is  given  rest,  medium  surrounding  temperature,  and  relatively 
equal  proteid  condition.  This  ride  not  only  applies  to  adult  human 
beings  but  also  to  children  and  growing  individuals  (Rubner,  Oppbn- 
HEIMBB).  The  surface  is  the  essential  factor  in  determining  the  extent  of 
exchange  of  energy.  In  order  to  show  this  we  will  give  here,  from  a  work 
of  Rubner,4  the  figures  representing  the  quantity  of  heat  in  calories  for 
1  square  meter  of  surface  for  twenty-four  hours. 


1  Skand.   Arch.   f.   Physiol.,  6. 

2  In  regard  to  metabolism  and  its  relationship  to  the  phases  of  sexual  life  and 
especially  under  the  influence  of  menstruation  and  pregnancy,  see  the  investigations 
of  A.  Ver  Eecke  (Bull.  acad.  roy.  de  med.  de  Belgique,  1897  and  1901,  and  Maiv's 
Jahresber.,  30  and  31). 

3  Rubner,  Zeitschr.  f.  Biologic,  19  and  81;   Richet,  Arch,  de  Physiol.  (5),  8. 

4  Rubner,  Ernahrung  im  Knabenalter,  45;  E.  Voit,  Zeitschr.  f.  Biologie,  41;  Oppen- 
heimer,  ibid.,  42. 


654  METABOLISM  WITH  VARIOUS  FOODS. 

Adult,  medium  diet,  rest 1189  Calories 

' '  "     medium  work 1399 

Suckling 1221 

Child  with  medium  diet 1447 

Aged  men  and  women 1099 

Women 1044 

The  variation  in  the  calorific  values  1  found  by  many  investigators, 
which  is  sometimes  not  very  small,  speaks  for  the  fact  that  the  surface 
rule  is  not  alone  decisive  for  the  exchange  of  material  in  resting  animals. 
Still  it  is  generally  considered  that  it  is  most  important  in  this  regard. 

The  more  active  metabolism  in  young  individuals  is  apparent  when 
we  measure  the  gaseous  exchange  as  well  as  the  excretion  of  nitrogen. 
As  example  of  the  elimination  of  urea  in  children  the  following  results  of 
Camerer  2  are  of  value: 

Age.  Weight  of  Body  in  Kilos.      Per   ^rea  in  Grainy  ^ 

1£  years 10.80 

3       "     


5 

7 

9 

12* 
15 


10.80 

12.10 

1.35 

13.30 

11.10 

0.90 

16.20 

12.37 

0.76 

18.80 

14.05 

0.75 

25.10 

17.27 

0.69 

32.60 

17.79 

0.54 

35.70 

17.78 

0.50 

In  adults  weighing  about  70  kilos,  from  30  to  35  grams  of  urea  per  day  are 
eliminated,  or  0.5  gram  per  kilo.  At  about  fifteen  years  of  age  the  destruc- 
tion of  proteids  per  kilo  is  about  the  same  as  in  adults.  The  relatively 
greater  metabolism  of  proteids  in  young  individuals  is  explained  partly 
by  the  fact  that  the  metabolism  of  material  in  general  is  more  active  in 
young  animals,  and  partly  by  the  fact  that  young  animals  are,  as  a  rule, 
poorer  in  fat  than  those  full  grown. 

According  to  Tigerstedt  and  Sonden  the  greater  metabolism  in  young 
animals  depends  nevertheless  also  in  part  on  the  fact  that  in  these  indi- 
viduals the  decomposition  in  itself  is  more  active  than  in  older  ones.  The 
period  of  growth  has  a  considerable  influence  on  the  extent  of  metabolism 
(in  man),  and  indeed  the  metabolism,  even  when  calculated  on  the  unit 
of  surface  of  body,  is  greater  in  youth  than  in  old  age.  This  view  is  strongly 
disputed  by  Rubner.  He  does  not  deny  that  differences  exist  between 
young  and  adult  individuals  which  may  be  considered  as  a  deviation  from 
the  above  rule;  still  these  differences  may,  according  to  Rubner,  be  de- 
pendent upon  the  work  performed,  the  food,  and  the  nutritive  condition. 
Magnus-Levy  and  Talk3  have  reported  observations  which  support  the 
views  of  Sonden  and  Tigerstedt. 

1  See  Magnus-Levy,  Pfliiger's  Arch.,  55;   Slowtzoff  (u.  Zuntz),  ibid.,  95. 

2  Zeitschr.  f.  Biologie,  16  and  20. 

3  Tigerstedt  and  Sond6n,  1.  c. ;  Rubner,  1.  c;  Magnus-Levy,  Arch.  f.  (Anat.  u.) 
Physiol.,  1899,  Suppl. 


RB8T  AND  WORK.  855 

As  the  metabolism  may  be  kepi  at  its  lowest  point  by  absolute  rest  of 

body  and  inactivity  of  the  intestinal  tract,  it  fa  manifesl  that  work  and 
th<-  ingestion  of  food  have  an  important  bearing  on  the  extent  of  metab- 
olism. 

Red  and  Work.  Daring  work  a  greater  quantity  of  chemical  energy  is 
converted  into  kinetic  energy,  i.e.,  the  metabolism  is  increased  more  or 
lese  on  account  of  work. 

As  explained  in  a  previous  chapter  (XI)  work,  according  to  the  gener- 
ally accepted  view,  has  no  material  influence  on  the  excretion  of  nitrogen. 
It  is  nevertheless  true  that  several  investigators  have  observed  in  certain 
an  increased  elimination  of  nitrogen;  but  these  observations  have 
been  explained  in  other  ways.  For  instance,  work  may,  when  it  is  con- 
nected with  violent  movements  of  the  body,  easily  cause  dyspnoea,  and  this 
last,  as  Frankel  l  has  shown,  may  occasion  an  increase  in  the  elimination 
of  nitrogen,  since  diminution  of  the  oxygen  supply  increases  the  proteid 
metabolism.  In  other  series  of  experiments  the  quantity  of  carbohydrates 
and  fats  in  the  food  was  not  sufficient;  the  supply  of  fat  in  the  body  was 
decreased  thereby,  and  the  destruction  of  proteids  was  correspondingly 
increased.  Other  conditions,  such  as  the  external  temperature  and  the 
weather,2  thirst,  and  drinking  of  water,  can  also  influence  the  excretion 
of  nitrogen.  According  to  the  generally  accepted  views  muscular  activity 
has  hardly  any  influence  on  the  metabolism  of  proteids. 

On  the  contrary,  work  has  a  very  considerable  influence  on  the  elimina- 
tion of  carbon  dioxide  and  the  consumption  of  oxygen.  This  action,  which 
irst  observed  by  Lavoisier,  has  recently  been  confirmed  by  many 
investigators.  Pettexkofer  and  Voit  3  have  made  investigations  on  a 
full-grown  man  as  to  the  metabolism  of  the  nitrogenous  as  well  as  of  the 
non-nitrogenous  bodies  during  rest  and  work,  partly  while  fasting  and 
partly  on  a  mixed  diet.  The  experiments  were  made  on  a  full-grown 
man  weighing  70  kilos.     The  results  are  contained  in  the  following  table: 

Consumption  of 
±Tote"  " 

*-*«■  -J3SL\    ft 

Mixed  diet  .|  »•*•_• 

In  these  cases  work  did  not  seem  to  have  any  influence  on  the  destruc- 
tion of  proteids.  while  the  gas  exchange  was  considerably  increased. 
Zuxtz  and  his  pupils  4  have  made  very  important  investigations  into 


'roteids. 

Fat.    Carbohydrates. 

C02  Eliminated 

O  Consumed 

78 

209 

716 

761 

75 

380 

1187 

1071 

137 

72             352 

912 

831 

137 

173             352 

1209 

980 

1  Yirchow's  Arch.,  07  and  71. 

Zuntz  and  Schumburg,  Arch.  f.  (Anat.  u.)  Physiol.,  1895. 
s  Zeitschr.  f.  Biologie,  2. 

*  See   the   works   of   Zuntz   and   Lehmann,    Italy's   Jahresber.,   19;     Katzenstein, 
Pfluger's  Arch.,  49;     Loewy,  ibid.;    Zuntz,  ibid.,  68,  and  especially  the  large  vrork 


656  METABOLISM  WITH  VARIOUS  FOODS. 

the  extent  of  the  exchange  of  gas  as  a  measure  of  metabolism  during  work 
and  caused  by  work.  These  investigations  not  only  show  the  important 
influence  of  muscular  work  on  the  decomposition  of  material,  but  they 
also  indicate  in  a  very  instructive  way  the  relationship  between  the  extent 
of  metabolism  of  material  and  useful  work  of  various  kinds.  We  can  only 
refer  to  these  important  investigations,  which  are  of  special  physiological 
interest. 

The  action  of  muscular  work  on  the  gas  exchange  does  not  alone  appear 
with  hard  work.  From  the  researches  of  Speck  and  others  we  learn  that 
even  very  small,  apparently  quite  unessential  movements  may  increase 
the  production  of  carbon  dioxide  to  such  an  extent  that  by  not  observing 
these,  as  in  numerous  older  experiments,  very  considerable  errors  may 
creep  in.  Johansson  1  has  also  made  experiments  upon  himself,  and  finds 
that  on  the  production  of  as  complete  a  muscular  inactivity  as  possible 
the  ordinary  amount  of  carbon  dioxide  (31.2  grams  per  hour  at  rest  in 
the  ordinary  sense)  may  be  reduced  nearly  one  third,  or  to  an  average  of 
22  grams  per  hour. 

The  quantity  of  carbon  dioxide  eliminated  during  a  working  period  is 
uniformly  greater  than  the  quantity  of  oxygen  taken  up  at  the  same 
time,  and  hence  a  raising  of  the  respiratory  quotient  was  usually  con- 
sidered as  caused  by  work.  This  rise  does  not  seem  to  be  based  upon  the 
character  of  chemical  processes  going  on  during  work,  as  we  have  a  series  of 
experiments  made  by  Zuntz  and  his  collaborators,  Lehmann,  Kat- 
zenstein  and  Hagemann,2  in  which  the  respiratory  quotient  remained 
almost  wholly  unchanged  in  spite  of  work.  According  to  Loewy  3  the 
combustion  processes  in  the  animal  body  go  on  in  the  same  way  in  work 
as  in  rest,  and  a  raising  of  the  respiratory  quotient  (irrespective  of  the 
transient  change  in  the  respiratory  mechanism)  takes  place  only  with 
insufficient  supply  of  oxygen  to  the  muscles,  as  in  continuous  fatiguing 
work  or  excessive  muscular  activity  for  a  brief  period,  also  with  local  lack 
of  oxygen  caused  by  excessive  work  of  certain  groups  of  muscles.  This 
varying  condition  of  the  respiratory  quotient  has  been  explained  by  Kat- 
zenstein  by  the  statement  that  during  work  two  kinds  of  chemical 
processes  act  side  by  side.  The  one  depends  upon  the  work  which  is  con- 
nected with  the  production  of  carbon  dioxide  also  in  the  absence  of  free 
oxygen,  while  the  other  brings  about  the  regeneration  which  takes  place 

"Untersuch.  iiber  den  Stoffwechsel  des  Pferdes  bei  Ruhe  und  Arbeit,"  Zuntz  and 
Hagemann,  Berlin,  1898,  which  also  contains  a  bibliography.  Zuntz  and  Slowtzoff, 
Pfl tiger's  Arch.,  95;   Zuntz,  ibid. 

^ord.  Med.  Arkiv.  Festband,  1897;  also  Maly's  Jahresber.,  27;  Speck,  "Physiol, 
des  menschl.  Athmens,"  Leipzig,  1892. 

2  See  foot-note  4,  page  G55. 

3  Pfluger's  Arch.,  49. 


ACTION  OF   THE  EXTERNAL   TEMPERATURE.  657 

by  the  taking  up  of  oxygen.  When  these  two  chief  kinds  of  chemical 
processes  make  the  same  progress  the  respiratory  quotient  remains  un- 
changed during  work;  if  by  hard  work  the  decomposition  is  increased  as 
compared  with  the  regeneration,  then  a  raising  of  the  respiratory  quotient 
takes  place.  If,  on  the  contrary,  moderate  work  is  continued  and  per- 
formed in  a  way  so  that  irregularities  and  occasional  changes  in  the  circu- 
lation and  respiration  arc  excluded  or  are  without  importance,  then  the 
respiratory  quotient  may  correspondingly  remain  the  same  during  work 
as  in  rest.  Its  extent  is  thereby  in  the  first  place  determined  by  the  nutri- 
tive material  at  its  disposal  (Zuntz  and  his  pupils). 

The  theory  of  Loewy  and  Zuntz  that  the  raising  of  the  respiratory  quotient 
during  work  is  to  be  explained  by  an  insufficient  supply  of  oxygen  is  opposed 
by  Laulanie.1  He  has  observed  the  reverse,  namely,  a  diminution  in  the  respira- 
tory quotient  during  continuous  excessive  work,  and  this  is  not  reconcilable  with 
the  above  statements.  According  to  Laulanie,  who  considers  sugar  as  the  source 
of  muscular  energy,  the  rise  in  the  respiratory  quotient  is  due  to  an  increased 
combustion  of  sugar.  The  diminution  of  the  same  he  explains  by  a  re-formation 
of  sugar  from  fat  which  takes  place  at  the  same  time  and  is  accompanied  by 
an  increased  consumption  of  oxygen. 

In  sleep  metabolism  decreases  as  compared  with  that  during  waking, 
and  the  most  essential  reason  for  this  is  the  muscular  inactivity  during 
sleep.  The  investigations  of  Rubner  upon  a  dog,  and  of  Johansson  2 
upon  human  beings,  teach  us  that  if  the  muscular  work  is  eliminated  the 
metabolism  during  waking  is  not  greater  than  in  sleep. 

The  action  of  light  also  stands  in  close  connection  with  the  question  of 
the  action  of  muscular  work.  It  seems  positively  proved  that  metabolism 
in  increased  under  the  influence  of  light.  Most  investigators,  such  as 
Speck,  Loeb,  and  Ewald,3  consider  that  this  increase  is  due  to  the  move- 
ments caused  by  the  light  or  an  increased  muscle  tonus.  Fubini  and  Bexi- 
dicexti  4  assume  that  the  increase  in  metabolism  due  to  light  is  independ- 
ent of  the  movements.  They  base  this  assumption  on  experiments  made 
on  hibernating  animals. 

Mental  activity  does  not  seem  to  have  any  influence  on  metabolism 
according  to  the  means  at  hand  for  studying  this  influence. 

Action  of  the  External  Temperature.  In  cold-blooded  animals  the  pro- 
duction of  carbon  dioxide  increases  and  decreases  with  the  rise  and  fall  of 
the  surrounding  temperature.  In  warm-blooded  animals  this  condition  is 
different.  By  the  investigations  of  Ludwig  and  Saxders-Ezn,  Pfluger 
and  his  pupils,  and  Duke  Charles  Theodore  of  Bavaria  and  others,5  it 

1  Arch,  de  Physiol.  (5),  8,  572. 

3  Rubner,  Ludwig-Festschr. ,  1887;  Loewy,  Berl.  klin.  Wochenschr.,  1891,  434; 
Johansson,  Skand.  Arch.  f.  Physiol.,  S. 

3  Speck,  1.  c;   Loeb,  Pfliiger's  Arch.,  42;   Ewald,  Journ.  of  Physiol.,  13. 

4  Cited  from  Maly's  Jahresber.,  22,  395. 

5  The  pertinent  literature  may  be  found  cited  by  Voit  in  Hermann's  Handbuch, 
C>.  and  also  by  Speck,  1.  c. 


658  METABOLISM  WITH  VARIOUS  FOODS. 

has  been  demonstrated  that  in  "warm-blooded  animals  the  change  in  the 
external  temperature  has  different  results  according  as  the  animal's  own 
heat  remains  the  same  or  changes.  If  the  temperature  of  the  animal  sinks, 
the  elimination  of  carbon  dioxide  decreases;  if  the  temperature  rises,  the 
elimination  of  C02  increases.  If,  on  the  contrary,  the  temperature  of  the 
body  remains  unchanged,  then  the  elimination  of  carbon  dioxide  increases 
with  a  lower  and  decreases  with  a  higher  external  temperature.  The 
statements  on  this  subject  are  somewhat  disputed  and  cases  have  been 
observed  where  in  warm-blooded  animals  the  metabolism  rises  on  cooling 
and  lowering  the  body  temperature,  while  warming  and  raising  the  body 
temperature  produces  a  diminution  (Krarup  1). 

The  increase  in  metabolism  produced  by  a  lowering  of  the  external 
temperature  is  explained,  according  to  Pfluger  and  Zuntz,  by  the  state- 
ment that  the  low  temperature,  by  exciting  a  reflex  action  in  the  sensitive 
nerves  of  the  skin,  causes  an  increased  metabolism  in  the  muscles  with  an 
increased  production  of  heat,  affecting  the  temperature  of  the  body,  while 
with  a  higher  external  temperature  the  reverse  takes  place.  The  experi- 
ments made  upon  animals  are  somewhat  uncertain  for  several  reasons,  but 
the  determinations  of  the  oxygen  absorption,  as  well  as  the  elimination  of 
C02,  made  by  Speck,  Loewy,  and  Johansson  2  in  human  beings,  have 
showm  that  cold  does  not  produce  any  essential  increase  in  the  metabolism 
of  man.  The  irritation  caused  by  cold  may  renexly  cause  a  forced  respira- 
tion with  an  action  on  the  gas  exchange,  and  weak  reflex  muscular  move- 
ments, such  as  shivering,  trembling,  etc.,  may  cause  an  insignificant  in- 
crease in  the  elimination  of  carbon  dioxide;  in  complete  muscular  inactivity 
cold  seems  to  cause  no  increased  absorption  of  oxygen  or  increased  meta- 
bolism. Eykman  's  3  experiments  upon  inhabitants  of  the  tropics  also  show 
the  same  result,  namely,  that  in  human  beings  no  appreciable  heat  regula- 
tion occurs. 

Metabolism  is  increased  by  the  ingestion  of  food,  and  Zuntz  4  has  cal- 
culated that  in  man  the  consumption  of  oxygen  is  raised  on  an  average 
15  per  cent  above  the  amount  during  rest  for  about  six  hours  after  taking  a 
moderately  hearty  meal.  This  increase  in  the  metabolism  is  caused, 
according  to  the  generally  accepted  view,  probably  only  by  the  increased 
work  of  the  digestive  apparatus  on  the  partaking  of  food.  Rjasantzefp 
has  shown  that  the  extent  of  nitrogen  elimination  is  proportional  to  the 


1  J.  C.  Krarup,  Den  omgifvende  temperaturs  indflydeke,  etc.,  Inaug.-Diss.  ,Kjoben- 
havn,  1902.  See  also*  Falloise,  Maly's  Jahresber.,  31;  Predteschensky,  ibid;  Rubner, 
Arch.  f.  Hygiene,  38. 

2  Speck,  1.  c;  Loewy,  Pfliiger's  Arch.,  46;  Johansson,  Skand.  Arch.  f.  Physiol.,  7. 
3Virchow's  Arch.,  133,  and  Pfliiger's  Arch.,  64. 

4  Zuntz  and  Levy,  "  Beitrag  zur  Kenntniss  d.  Verdaulichkeit,  etc.,  des  Brodes," 
Pfliiger's  Arch.,  49;  Magnus-Levy,  ibid.,  55;  Koraen,  Skand.  Arch.  f.  Physiol  ,  11; 
Johansson  and  Koraen,  ibid.,  13. 


THE  NECESSITY  OF  FOOD  BY  MAN.  669 

intensity  of  the  digestive  work.  It  also  follows  from  the  works  of  Magnus- 
I.i;\  v,  KoBAEN  and  JOHANSSON  l  that  the  proteids  and  to  a  lesser  extent  the 
carbohydrates  even  by  themselves  produce  a  rise  in  metabolism  which 
does  not  seem  to  be  true  for  the  fats. 

VI.  The  Necessity  of  Food  by  Man  under  Various  Conditions. 

Various  attempts  have  been  made  to  determine  the  daily  quantity  of 
organic  food  needed  by  man.  Certain  investigators  have  calculated,  from 
the  total  consumption  of  food  by  a  large  number  of  similarly  fed  individuals — 
soldiers,  sailors,  laborers,  etc. — the  average  quantity  of  foodstuffs  required 
per  head.  Others  have  calculated  the  daily  demand  of  food  from  the  quan- 
tity of  carbon  and  nitrogen  in  the  excreta  or  calculated  it  from  the  exchange 
of  force  of  the  person  experimented  upon.  Others,  again,  have  calculated 
the  quantity  of  nutritive  material  in  a  diet  by  which  an  equilibrium  was 
maintained  in  the  individual  for  one  or  several  days  between  the  consump- 
tion and  the  elimination  of  carbon  and  nitrogen.  Lastly,  still  others  have 
quantitatively  determined  during  a  period  of  several  days  the  organic 
foodstuffs  consumed  daily  by  persons  of  various  occupations  who  chose 
their  own  food,  by  which  they  were  well  nourished  and  rendered  fully  capa- 
ble of  labor. 

Among  these  methods  a  few  are  not  quite  free  from  objection,  and  others 
have  not  as  yet  been  tried  on  a  sufficiently  large  scale.  Nevertheless  the 
experiments  collected  thus  far  serve,  partly  because  of  their  number  and 
partly  because  the  methods  correct  and  control  one  another,  as  a  good 
starting-point  in  determining  the  diet  of  various  classes  and  similar  ques- 
tions. 

If  the  quantity  of  foodstuffs  taken  daily  be  converted  into  calories 
produced  during  physiological  combustion,  we  then  obtain  some  idea  of 
the  sum  of  the  chemical  energy  which  under  varying  conditions  is  intro- 
duced into  the  body.  It  must  not  be  forgotten  that  the  food  is  never 
completely  absorbed,  and  that  undigested  or  unabsorbed  residues  are 
always  expelled  from  the  body  with  the  fasces.  The  gross  results  of  calories 
calculated  from  the  food  taken  must  therefore,  according  to  Rubner,  be 
diminished  by  at  least  8  per  cent.  This  figure  is  true  at  least  when  the  human 
being  partakes  of  a  mixed  diet  of  about  60  per  cent  of  the  proteids  as  ani- 
mal and  about  40  per  cent  of  the  proteids  as  vegetable  foodstuffs.  With 
more  one-sided  food,  especially  when  this  is  rich  in  undigestible  cellulose, 
a  much  larger  quantity  must  be  subtracted. 

The  following  summary  contains  a  few  examples  of  the  quantity  of 
food  which  is  consumed  by  individuals  of  various  classes  of  people  under 
different  conditions.  In  the  last  column  we  also  find  the  quantity  of  living 
force  which  corresponds  to  the  quantity  of  food  in  question,   calculated 

1  See  foot-note  4,  page  658. 


'-'--  METABOLISM  WITH   VARIOUS  FOODS. 

as  calories,  with  tiie  above-stated  correction.     The  calories  are  therefore 
net  results,  while  the  figures  for  the  nutritive  bodies  are  gross  results. 

Protefck.    Wat        -:     j"    Odoriea.    Authority. 


eaoe. 119  40  529  27*4     Platfair.1 

rice. 117  35  447  2424     Hildesheim. 

146  46  504  2852 

130  40   .      5-50  2903     Moleschott. 

137  72  352  2458     Pettexeofze  and  Vorr. 

(40  years).  131  68  494  2835     Forsxz?..: 

a... 127  89  362  2602 

134  102  292  247 . 


L:-    .:-    \-      -  r-                   V-_\  >f  .__  _:.: 

E^siiii.  sni-h 1".  "1  ■:■:■:  \'Y.  ?:_<l— air. 

r:^:- 2^5  ;";  93  21 

Bavarian  wood-chopper. .    135  208  876  5589  Ltebig. 

Laborer  in  Silesia. 80  16  .552  251S  Meevert.3 

Seamstress  in  London ...  .     54  29  292  1688  Platfaie. 

Swedish  laborer. 134  79  485  3019  Hultgrex  and  Landergren'.* 

Japanese  student 83  14  622  2779  Eijkmax.5 

55  6  394  1744  Tawara.5 


It  k  evident  ::-at  person-  \:  essentially  different  weight  of  body  who 
lhre  under  unequal  external  conditions  must  need  essentially  different  food. 
^Iso  to  be  expected  (and  this  is  confirmed  by  the  table)  that  not  only 
the  absolute  quantity  of  food  consumed  by  various  persons,  but  also  the 
relative  proportion  of  the  various  organic  nutritive  substances,  shows  con- 
siderable variation.  Results  for  the  daily  need  of  human  beings  in  general 
cannot  be  given.  Tor  certain  classes  such  as  soldiers,  laborers,  etc., 
results  may  be  given  which  are  valuable  for  the  calculation  of  the  daily 
rations. 

Based  on  extensive  investigations  and  a  very  wide  experience,  Voit  has 
proposed  the  following  average  quantities  for  the  daily  diet  of  adults: 

~ — =:';•  Fat.  Ci-V„hydrates.     Calories. 

J.:  rr_e:i. 11*  grams         56  grams  500  grams  2810 

But  it  should  be  remarked  that  these  data  relate  to  a  man  weighing 
70  i     ~r  kilos  and  wh  o  i  raged  daily  for  ten  hours  in  not  too  f atigu- 

The  quantity  of  food  required  by  a  woman  engaged  in  moderate  work 
is  about  four  fifths  that  of  a  laboring  man,  and  we  may  consider  the  fol- 
lowing as  a  daily  diet  with  moderate  work: 

•  -  -.'hydrates.     Calories. 
-  Tomen. 94  grams         45  grams         400  grams         2240 

1  In  regard  to  the  older  researches  cited  in  this  table  we  refer  the  reader  to  Voit  in 
Hermann's  Handbuch.  6,  519. 

2  Ibid.,  and  Zeitschr.  L  Bkiqgj 

*  Armee-  raid  Volksernahrung,  Berlin,  1SS0. 

-.tersuchung  uber  die  Ernahrung  schwedischer  Arbeiter  bei  frei  gewahlter  Kost. 
Stockholm,  1891.     Maly's  Jahresber.,  21 

1  Cited  from  Kellner  and  Mori  in  Zeitschr.  f.  Biologie.  2'j 


THE  NECESSITY  OF  FOOD  BY  MAN.  661 

The  proportion  of  fat  to  carbohydrates  is  here  as  1 : 8-9.  Such  a  pro- 
portion occurs  often  in  the  food  of  the  poorer  classes  which  live  chiefly 
upon  the  cheap  and  voluminous  vegetable  food  while  this  ratio  in  the  food 
of  wealthier  persons  is  1 :  3-4.  It  would  be  desirable  if  in  the  above  rations 
the  fat  was  increased  at  the  expense  of  the  carbohydrates,  but  unfortu- 
nately on  account  of  the  high  price  of  fat  such  a  modification  cannot  always 
be  made. 

In  examining  the  above  numbers  for  the  daily  rations  it  must  not  be 
forgotten  that  the  figures  for  the  various  foodstuffs  are  gross  results.  They 
consequently  represent  the  quantity  of  these  which  must  be  taken  in,  and 
not  those  which  are  really  absorbed.  The  figures  for  the  calories  are,  on 
the  contrary,  net  result-. 

The  various  foods  are,  as  is  well  known,  not  equally  digested  and 
absorbed,  and  in  general  the  vegetable  foods  are  less  completely  consumed 
than  animal  foods.  This  is  especially  true  of  the  proteids.  When,  there- 
fore, Voit,  as  above  stated,  calculates  the  daily  quantity  of  proteids  needed 
by  a  laborer  as  118  grams,  he  starts  with  the  supposition  that  the  diet  is  a 
mixed  animal  and  vegetable  one,,  and  also  that  of  the  above  118  grams 
about  105  grams  are  absorbed.  The  results  obtained  by  Pfluger  and 
his  pupils  Bohlaxd  and  Bleibtreu  1  on  the  extent  of  the  metabolism  of 
proteids  in  man  with  an  optional  and  sufficient  diet  correspond  well  with 
the  above  figures,  when  the  unequal  weight  of  body  of  the  various  persons 
experimented  upon  is  sufficiently  considered. 

As  a  rule,  the  more  exclusively  a  vegetable  food  is  employed,  the 
smaller  is  the  quantity  of  proteids  in  the  same.  The  strictly  vegetable  diet 
of  certain  people,  as  that  of  the  Japanese  and  of  the  so-called  vegetarians, 
is  therefore  a  proof  that,  if  the  quantity  of  food  be  sufficient,  a  person  may 
exist  on  considerably  smaller  quantities  of  proteids  than  Voit  suggests. 
It  follows  from  the  investigations  of  Hirschfeld.  Kumagawa  and  Klem- 
perer.  Sivex,  and  others  (see  page  647)  that  a  nearly  complete  or  indeed 
a  complete  nitrogenous  equilibrium  may  be  attained  by  the  sufficient 
administration  of  non-nitrogenous  nutritive  bodies  with  relatively  vary 
small  quantities  of  proteids. 

If  we  bear  in  mind  that  the  food  of  people  of  different  countries  varies 
greatly,  and  that  the  individual  also  takes  essentially  different  nourish- 
ment according  to  the  external  conditions  of  living  and  the  influence  of 
climate,  it  is  not  remarkable  that  a  person  accustomed  to  a  mixed  diet 
can  exist  for  some  time  on  a  strictly  vegetable  diet  deficient  in  proteids. 
No  one  doubts  the  ability  of  man  to  adapt  himself  to  a  heterogeneously 
composed  diet  when  this  is  not  too  difficult  of  digestion  and  is  sufficient 
in  quantity:  also  we  cannot  deny  that  it  is  possible  for  a  man  to  exist 
also  for  a  long  time  with  smaller  amounts  of  proteid  than  Voit  suggests, 

1  Bohland,  Pfluger  "s  Arch..  3«5:    Bleibtreu,  ibid.,  3S. 


662  METABOLISM  WITH  VARIOUS  FOODS. 

namely  118  grams.  Thus  0.  Neumann1  experimented  on  himself  during 
746  days  in  three  series  of  experiments  and  his  diet  consisted  of  74.2  grams 
proteid,  117  grams  fat,  and  213  grams  carbohydrates  (  =  2367  gross  calories, 
with  a  weight  of  70  kilos  and  with  ordinary  laboratory  work).  These 
figures  cannot  be  compared  with  those  obtained  by  Voit's  worker,  weigh- 
ing 70  kilos,  whose  work  was  harder  than  a  tailor's  and  easier  than  a  black- 
smith's; for  example,  the  work  of  a  mason,  carpenter,  or  cabinet-maker. 
The  observations  made  thus  far  on  a  lower  proteid  consumption  give  no 
reason  for  essentially  changing  Voit's  figure.  Although  man  ma}*  be 
satisfied  under  certain  circumstances  with  a  smaller  quantity  of  proteid 
than  that  calculated  by  Voit,  still  it  does  not  follow  that  such  a  diet  is 
also  the  most  serviceable.  Voit's  figures  are  only  given  for  certain  cases 
or  certain  categories  of  human  beings.  It  is  apparent  that  other  figures 
must  be  taken  for  other  cases,  and  it  is  evident  that  the  daily  ration  given 
by  Voit  as  necessary  for  a  laborer  must  be  altered  slightly  for  other  coun- 
tries because  of  the  existing  conditions  in  middle  Europe,  where  Voit 
made  his  investigations.  The  numerous  compilations  (of  Atwater  and 
others  2)  on  the  diet  of  different  families  in  America  have  given  the  figures 
97-113  grams  proteid  for  a  man,  and  the  very  careful  investigations  of 
Hultgren  and  Landergren  have  shown  that  the  laborer  in  Sweden 
with  moderate  work  and  an  average  body  weight  of  70.3  kilos,  with 
optional  diet,  partakes  134  grams  proteid,  79  grams  fat,  and  522  grams 
carbohydrates.  The  quantity  of  proteid  is  here  greater  than  is  necessary, 
according  to  Voit.  On  the  other  hand  Lapicque  3  found  67  grams  pro- 
teid for  Abyssinians  and  81  grams  for  Malaysians  (per  body  weight  of  70 
kilos),  materially  lower  figures. 

If  we  compare  the  figures  on  page  660  with  the  average  figures  proposed 
by  Voit  for  the  daily  diet  of  a  laborer,  it  would  seem  at  the  first  glance  as 
if  the  food  consumed  in  certain  cases  was  considerably  in  excess  of  the  need, 
while  in  other  cases,  as,  for  instance,  that  of  a  seamstress  in  London,  it  was 
entirely  insufficient.  A  positive  conclusion  cannot,  therefore,  be  drawn  if 
we  do  not  know  the  weight  of  the  body,  as  well  as  the  labor  performed  by 
the  person,  and  also  the  conditions  of  living.  It  is  certainty  true  that  the 
amount  of  nutriment  required  by  the  body  is  not  directly  proportional  to 
the  body  weight,  for  a  small  body  consumes  relatively  more  substance 
than  a  larger  one,  and  varying  quantities  of  fat  may  also  cause  a  difference ; 
but  a  large  body,  which  must  maintain  a  greater  quantity,  consumes  an 

1  Arch.  f.  Hygiene,  45. 

2  Atwater,  Report  of  the  Storrs  Agric.  Expt.  Station,  Conn.,  1891-1895  and  1896; 
also  Nutrition  Investigations  at  the  University  of  Tennessee,  1896  and  1897;  U.  S. 
Dept.  of  Agriculture,  Bull.  53,  1898.  See  also  Atwater  and  Bryant,  ibid.,  Bull.  "5; 
Jaffa,  ibid.,  84;   Grindley,  Sammis,  and  others,  ibid.,  91. 

3  Hultgren  and  Landergren,  1.  c. ;   Lapicque,  Arch,  de  Physiol.  (5),  6. 


THE  NECESSITY  FOR  FOOD  BY  MAN.  663 

absolutely  greater  quantity  of  substance  than  a  small  one,  and  in  estimat- 
ing the  nutritive  need  one  must  also  always  consider  the  weight  of  the  body. 
According  to  Voit,  the  diet  for  a  laborer  with  70  kilos  body  weight  requires 
40  calories  for  each  kilo.  Ekholm  l  calculates,  basing  it  upon  his  experi- 
ments, that  for  a  man  weighing  70  kilos,  busied  with  reading  and  writing, 
the  net  calories  are  2450  and  the  gross  calories  2700,  or  35  and  38.5  calories 
per  kilo.  The  minimum  figure  for  metabolism  during  sleep  and  in  as 
complete  rest  as  possible  has  been  found  by  Sonden,  Tigerstedt  and 
Johansson  2  to  be  24-25  calories. 

As  several  times  stated  above,  the  demands  of  the  body  for  nourishment 
vary  with  different  conditions  of  the  body.  Among  these  conditions  two 
are  especially  important,  namely,  work  and  rest. 

In  a  previous  chapter,  in  which  muscular  labor  was  spoken  of,  it  was 
seen  that  all  foodstuffs  have  nearly  the  same  power  of  serving  as  a  source 
for  muscular  work,  and  that  the  muscles,  it  seems,  select  that  foodstuff 
which  is  supplied  to  them  in  the  greatest  quantity.  As  a  natural  sequence 
it  is  to  be  expected  that  muscular  activity  requires  indeed  an  increased  sup- 
ply of  foodstuffs,  but  no  essential  change  in  the  relation  of  the  same,  as 
compared  to  rest. 

Still  this  does  not  seem  to  hold  true  in  daily  experience.  It  is  a  well- 
known  fact  that  hard-working  individuals — men  and  animals — require  a 
greater  quantity  of  proteids  in  the  food  than  less  active  ones.  This  contra- 
diction is,  however,  only  apparent,  and  it  depends,  as  Voit  has  shown,  upon 
the  fact  that  individuals  used  to  violent  work  are  more  muscular.  For 
this  reason  a  person  performing  severe  muscular  labor  requires  food  con- 
taining a  larger  proportion  of  proteids  than  an  individual  whose  occupation 
demands  less  violent  exertion.  Another  fact  is  that  the  diet  rich  in  pro- 
teids is  often  concentrated  and  less  bulky,  and  also  that  in  many  cases  of 
training  a  diet  containing  as  little  fat  as  possible  is  selected. 

If  we  compare  the  results  for  the  needs  of  food  in  work  and  rest  which 
are  obtained  under  conditions  which  can  be  readily  controlled,  it  is  found 
that  the  above  statements  are  confirmed  in  general.  As  example  of  this 
the  following  table  gives  the  rations  of  soldiers  in  peace  and  in  the  field 
and  the  average  figures  from  the  detailed  data  of  various  countries.3 

A.  Peace  Ration.  B.  War  Ration. 

Proteids.  Fat.  Carbohydrates.  Proteids.  Fat.  Carbohydrates. 

Minimum 108  22             504  126  38             484 

Maximum 165  97             731  197  95             688 

Mean 130  40             551  146  59             557 

1  Skand.  Arch.  f.  Physiol.,  11. 

'Sonden  and  Tigerstedt,  Skand.  Arch.  f.  Physiol.,  6;  Johansson,  ibid.,  7;  Tiger- 
stedt, Nord.  Med.  Arkiv.  Festband,  1897. 

1  Germany,  Austria,  Switzerland,  France,  Italy,  Russia,  and  the  United  States.  It  is 
not  known  hy  the  author  whether  these  figures  have  been  changed  in  the  last  few 
years  in  the  various  countries,  and  hence  whether  they  must  be  modified  or  not. 


664  METABOLISM  WITH  VARIOUS  FOODS. 

The  following  figures  for  the  daily  ration  are  obtained  from  the  above 
averages: 

Proteids.  Fat.        Carbohydrates.      Calories. 

In  peace 130  40  551  2900 

In  war 146  59  557  3250 

If  we  calculate  the  fat  in  its  equivalent  quantity  of  starch,  then  the 
relation  of  the  proteids  to  the  non-nitrogenous  foods  isj 

In  peace 1  :  4.97 

In  war 1  :  4.79 

The  relation  in  both  cases  is  nearly  the  same.  Similar  results  are 
obtained  when  we  start  with  Voit  's  figures  for  a  soldier  in  manoeuvre  A 
(hard  work)  and  B  (strenuous  work)  in  war. 


Proteids. 

Fat. 

Carbohydrates. 

Calories. 

.   135 

80 

500 

3013 

.    145 

100 

500 

3218 

The  relation  here,  when  the  fat  is  recalculated  as  starch,  in  both  cases  is 
the  same,  or  equal  to  1:5. 

If  we  calculate  that  portion  of  the  total  calories  supplied,  which  falls  to 
each  group  of  the  foodstuffs,  it  is  found  that  16-19  per  cent  comes  from  the 
proteid  in  rest  as  well  as  with  medium  and  strenuous  work.  For  the  fat 
and  the  carbohydrates  the  variations  are  greater;  the  chief  quantity  of 
calories  comes  from  the  carbohydrates.  Of  the  total  calories  16-30 
per  cent  comes  from  the  fat  and  50-67  per  cent  from  the  carbohydrates. 

The  importance  of  the  food-demand  for  working  individuals  is  shown 
by  the  figures  given  on  page  660  for  a  wood-chopper  in  Bavaria.  A  need 
of  more  than  4000  calories  occurs  only  seldom,  and  with  very  hard  work 
the  demand  may  rise  even  to  7000  calories  (Atwater  and  Bryant,  Jaffa  1). 

As  more  work  requires  an  increase  in  the  absolute  quantity  of  food,  so 
the  quantity  of  food  must  be  diminished  when  little  work  is  performed. 
The  question  as  to  how  far  this  can  be  done  is  of  importance  in  regard  to 
the  diet  in  prisons  and  poorhouses.  We  give  below  the  following  as  exam- 
ple of  such  diets: 

Proteids.  Fat.  Carbohydrates.  Calories. 

Prisoner  (not  working) .  .   87  22  305  1667     Schuster.2 

"         "         ..85  30  300  1709     Voit. 

Man  in  poorhouse 92  45  232  1985     Forster.3 

Woman  in  poorhouse.  . .  .   80  49  266  1725 

The  figures  given  by  Voit  are,  he  says,  the  lowest  reported  for  a  non- 


1  See  foot-note  2,  page  662. 

2  See  Voit,  Unterauchung  der  Kost.     Miinchen,  1877,  142.     See  also  Hirschfeld, 
Maly's  Jahresber.,  30. 

3  Ibid..  186. 


OBESITY   CURES.  865 

working  prisoner.     He  considers  the  following  as  the  lowest  diet  for  old 
Don-working  people: 

Proteids.  Fat.  Carbohydrates.      Calories. 

Men 90  40  350  2200 

Women 80  35  300  1733 

In  calculating  the  daily  diet  it  is  in  most  cases  sufficient  to  ascertain 
how  1 1 inch  of  the  various  foodstuffs  must  be  administered  to  the  body  in 
order  to  keep  it  in  the  proper  condition  to  perform  the  work  required  of 
it.  In  other  cases  it  may  be  a  question  of  improving  the  nutritive  con- 
dition of  the  body  by  properly  selected  food;  and  there  also  are  cases  in 
which  it  is  desired  to  diminish  the  mass  or  weight  of  the  body  by  an  insuf- 
ficient nutrition.  This  is  especially  the  case  in  obesity,  and  all  the  die- 
taries proposed  for  this  purpose  are  chiefly  starvation  cures  which  will  be 
shown  below  from  those  selected,  namely,  Harvey,  Ebstein  and  Oertel's 
cure. 

The  oldest  and  most  generally  known  diet  cure  for  corpulency  is  that  of 
Harvey,  which  is  ordinarily  called  the  Banting  method.  The  principle 
of  this  cure  consists  in  increasing,  as  far  as  possible,  the  consumption  of 
the  accumulated  fat  of  the  body  by  as  limited  a  supply  of  fat  a'nd  carbo- 
hydrates as  practicable  and  a  simultaneously  increased  supply  of  proteids. 
A  second  called  Ebstein 's  cure,  based  on  the  assumption  (not  correct) 
that  the  fat  of  the  food  is  not  accumulated  in  a  body  rich  in  fat,  but  is 
completely  burnt.  In  this  cure  large  quantities  of  fat  are  therefore  allowed 
in  the  food,  while  the  quantity  of  carbohydrates  is  diminished  very  mate- 
rially. The  third  cure,  called  Oertel's  *  cure,  is  based  on  the  correct 
view  that  a  certain  quantity  of  carbohydrates  has  no  greater  influence 
in  the  accumulation  of  fat  than  the  isodynamic  quantities  of  fat.  In  this 
cure,  therefore,  carbohydrates  as  well  as  fat  are  allowed,  provided  the 
total  quantity  of  the  same  is  not  so  great  as  to  hinder  the  decrease  in  the 
fatty  condition.  A  greatly  diminished  supply  of  water  is  also  one  of  the 
features  of  Oertel's  cure,  especially  in  certain  cases.  The  average  quan- 
tity of  the  various  nutritive  substances  supplied  to  the  body  in  these  three 
cures  is  as  follows,  and  we  give  also  for  comparison  in  the  same  table  Voit's 
diet  necessary  for  a  laborer. 

Proteids.  Fat.    Carbohydrates.  9^°"^ 

(gr< 

Harvey-Banting  's  cure 171  8  75  L083 

Eustein 's  cure 102  85  47  1396 

Oertel's      "     156  22  72  1140 

"    (max.) 170  44  114  1573 

Laborer,  according  to  Voit 118  56  500  3055 

1  Banting,  Letter  on  Corpulence.  London,  1864.  Ebstein,  Die  Fettliebigkeit  und 
ihre  Behandlung.  1882.  Oertel,  Handbuch  der  allg.  Therapie  der  Kreislaufstorungen. 
1884. 


666  METABOLISM  WITH  VARIOUS  FOODS. 

If  the  fat  in  all  cases  is  recalculated  in  starch,  then  the  proportion  of 
the  proteids  to  the  carbohydrates  is: 

Harvey- Banting's  cure 100  :    54 

Ebstein  's  cure 100  :  240 

Oertel's     "   100  :    80 

"    (max.) 100  :  129 

Laborer 100  :  530 

In  all  these  cures  for  corpulence  the  quantity  of  non-nitrogenous  bodies 
is  diminished  as  compared  with  the  proteids;  but  also  the  total  quantity 
of  food,  as  is  shown  by  the  number  of  calories,  is  considerably  diminished. 

Harvey-Banting's  cure  differs  from  the  others  in  a  relatively  very 
much  greater  quantity  of  proteids,  while  the  total  number  of  calories  in  it 
is  the  smallest.  On  this  account  this  cure  acts  very  quickly;  but  it  is 
therefore  also  more  dangerous  and  more  difficult  to  accomplish.  In  this 
regard  Ebstein 's  and  Oertel's  cures  (especially  Oertel's),  having  a 
greater  variation  in  the  selection  of  food,  are  better.  As  the  adipose 
tissue  has  a  proteid-sparing  action,  we  have  to  consider  in  using  these 
cures,  especially  Banting's,  that  the  destruction  of  proteids  in  the  body 
is  not  increased  in  the  adipose  tissue,  and  one  must  therefore  carefully  watch 
the  elimination  of  nitrogen  by  the  urine.  All  diet  cures  for  obesity  are 
moreover,  as  above  stated,  starvation  cures;  and  if  the  daily  quantity 
of  food  required  by  an  adult  man,  represented  as  calories,  is  in  round 
numbers  2500  calories  (according  to  the  average  figures  found  by  Forster 
in  the  case  of  a  physician),  then  one  immediately  sees  what  a  considerable 
part  of  its  own  mass  the  body  must  daily  give  up  in  the  above  cures.  This 
reminds  us  of  the  great  care  necessary  in  employing  them;  each  special 
case  should  be  conducted  with  regard  to  the  individuality,  the  weight  of 
the  body,  the  elimination  of  nitrogen  in  the  urine,  etc.,  etc.,  and  always 
under  strong  control,  and  only  by  a  physician,  never  by  a  layman.  A 
more  detailed  discussion  of  the  many  conditions  which  must  be  considered 
in  these  cases  does  not  enter  into  the  plan  and  scope  of  this  work. 


ANIMAL  FOODS. 
TABLE  I.— FOODS.1 


667 


I.  Animal  Foodstuffs. 


1000  Parts  contain 

1 

2 

3 

4 

5 

6 

a! 

is 

-  « 

C 

2 

0 « 

«j 

•SB 

-C 

a 

Oh 

£ 

*T3 

< 

£ 

* 

183 

166 

11 

640 

196 

98 

18 

688 

190 

120 

18 

672 

218 

115 

117 

550 

190 

80 

13 

717 

318 

65 

125 

492 

255 

365 

100 

280 

100 

660 

40 

130 

233 

11 

12 

744 

195 

93 

11 

701 

253 

14 

14 

719 

246 

31 

12 

711 

156 

141 

9 

544 

150 

167 

83 

15 

585 

150 

175 

93 

85 

480 

167 

190 

100 

100 

430 

180 

135 

332 

8 

437 

88 

160 

160 

10 

520 

150 

100 

460 

5 

365 

70 

120 

540 

60 

200 

80 

200 

300 

70 

340 

90 

89 

220 

6 

352 

333 

121 

67 

10 

469 

333 

128 

39 

11 

489 

333 

145 

14 

11 

580 

250 

100 

2 

8 

440 

450 

86 

1 

8 

455 

450 

82 

1 

6 

4(11 

450 

140 

140 

100 

280 

340 

116 

43 

107 

334 

400 

200 

108 

132 

460 

100 

246 

1 

178 

472 

100 

532 

5 

106 

257 

100 

665 

10 

59 

116 

150 

736 

7 

87 

170 

Relationship  of 
1:2  8. 


a.  Meat  without  Bones. 

Fat  beef 2 

Beef  (average  fat  ') 

Beef 2 

Corned  beef  (average  fat) 

Veal 

Horse,  salted  and  smoked. .  .  . 

Smoked  ham 

Pork,  salted  and  smoked  3.  .  . 
Meat  from  hare 

"        ."     chicken 

"         "     partridge 

"         "     wild  duck 

6.  Meat  with  Bones. 


Fat  beef  2 

Beef  (average  fat  *) 

Beef,  slightly  corned 

Beef,  thoroughly  corned 

Mutton,  very  fat 

"        average  fat 

Pork,  fresh,  fat 

"      corned,  fat 

Smoked  ham 

c.  Fishes. 

River  eel,  fresh,  entire 

Salmon,       "  "      

Anchovy,    "  "      

Flounder,    "  " 

River  perch,  fresh,  entire 

Torsk,  "  "     

Pike.  "  "     

Herrinp,  salted,  entire 

Anchovy,    "  "     

Salmon  (side"),  salted 

Kabeljau  (salted  haddock) 

Codfish  (dried  ling") 

(dried  torsk) 

Fish-meal  from  variety  of  Gadus 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 

100 

1(1(1 
1(11) 

100 
100 


90 

50 

63 

53 

42 

20 

143 

660 

5 

48 

6 

13 


90 

49 

53 

53 

246 

1Q0 

460 

450 

150 


246 

56 

31 

9 

2 

1 

1 

100 

37 

54 

1 

1 

1 

1 


1  The  results  in  the  following  tables  are  chiefly  compiled  from  the  summary  of  AllfftH  and  of 
KOmo.  We  here  designate  as  "waste"  that  part  of  the  foods  which  is  lost  in  the  preparation 
or  that  which  Is  not  used  by  the  body;  for  instance,  bones,  skin,  egg-shells,  and  the  cellulose 
vegetable  foods. 

=  Meat  such  as  is  ordinarily  sold  in  the  markets  in  Sweden. 

3  Pork,  chiefly  from  the  breast  and  belly,  such  as  occurs  in  the  rations  of  Swedish  soldiers. 


668 


FOOD   TABLES. 
TABLE  L— FOODS— (Continued). 


1000  Parts  contain 

Relationship  of 
1:2:3. 

i.  Animal  Foodstuffs. 

1 
Ph 

2 

3 

-   - 

4 

03 
< 

5 

u 

1 

6 

s 

1 

1 

:2 

:3 

d.  Inner  Organs  (Fresh). 

116 
196 
184 
163 
221 
150 

182 

190 

220 

7 

3 

304 

35 

35 

41 

37 

230 

334 

89 

106 

122 

160 

103 

123 

110 

92 

150 

88 

90 

115 

115 

114 

77 

80 

111 

110 

117 

140 

101 

70 

232 

220 

270 

103 
56 
92 

106 
38 

170 

2 

150 

160 
850 
990 

35 

7 

9 

257 

270 

66 

70 

93 

107 

307 

7 

17 
10 
11 
39 
10 

3 
17 
15 
20 
10 
14 
21 
10 
60 
60 
58 

7 
21 
15 
15 

11 

7 

50 
50 
38 
35 

40 

50 

456 

4 

5 

7 

676 
740 
768 
439 
550 
768 
688 
720 
725 
480 
514 
654 
720 
563 
660 
656 
770 
537 
530 
520 

11 

17 
10 
10 
13 
10 

9 

50 
55 
15 

175 

7 

7 

7 

6 

60 

50 

56 

8 

10 

13 

8 

18 

8 

3 

50 

17 

8 

18 

20 

15 

16 

11 

26 

7 

30 

20 

17 

2 

36 

25 

25 

770 

720 
714 
721 
728 
670 

807 

610 
565 
119 
7 
217 
873 
901 
905 
665 
400 
500 
329 
654 
756 
520 
875 

140 
120 
120 
130 
330 
131 
140 
110 
110 
400 
370 
140 
146 
130 
100 
140 
146 
137 
150 
125 

135 

26 

12 

6 

192 

5 

22 
20 
16 
17 
11 
48 

7 

100 

20 

28 

5 
37 
60 
45 

100 
100 
100 
100 
100 
100 

100 

100 
100 
100 
100 

100 

100 
100 
100 
100 
100 
100 
100 
100 
100 
100 

100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 

89 
28 
50 
65 
17 
113 

1 

•   79 

73 

12100 

33000 

100 
20 
22 

695 

117 
19 
79 
88 
88 

192 
7 

14 

11 

12 

26 

11 

3 

15 

13 

18 

14 

18 

19 

9 

51 

43 

57 

10 

9 

7 

6 

0 

0 

0 

Heart  and  lungs  of  mutton 

Yeal-kidney 

0 
0 

0 

Blood  from  various  animals  (av- 
erage results) 

0 

e.  Other  Animal  Foods. 
Variety  of  pork-sausage  (Mett- 

0 

Same  for  frying 

0 

Butter            

100 

0 

Cow's  milk  (full) 

143 

"         "     (skimmed) 

Buttermilk 

143 
93 

95 

17 

"       (poor)    

15 

Whey  cheese  (poor) 

512 

Hen's  egg,  entire 

4 

Yolk  of  egg 

4 
0 

White  of  egg.  .        

7 

2.  Vegetable  Foodstuffs. 

.549 

Wheat-flour  (fine) 

654 

Wheat-bran 

835 
292 

Wheat-bread  (fresh) 

625 

Macaroni    

853 

Rye  (grains) 

600 

Rve-flour 

626 

Rye-bread  (dry) 

634 

"        "      (fresh,  coarse) 

"      (fresh,  fine) 

Barley  (grains) 

623 
634 

589 

Scotch  barley 

654 

Oat  (grains) 

481 

"     (peeled) 

471 

Corn.  .                      

662 

Rice  (peeled  for  boiling)   

French  beans 

1100 
231 

Peas  (yellow  or  green,  dry).  .  .  . 
Flour  from  peas 

240 
192 

VEGETABLE  FOODS  AND  LIQUORS. 
TABLE  I.— FOODS—  (Continued). 


6(39 


1000  Parts  contain 

Relationship  of 

a.  Vegetable  Foodstuffs. 

1 

3.2 

s  a 

~  M 

2 
n 

3 

k 

b  u 

4 

A 

X 

< 

5 

c 
a 

"3 

6 

6 
a 

1 

:2 

3 

20 

14 

10 

25 

19 

27 

31 

14 

10 

12 

32 

219 

4 

5 

242 

140 

2 
2 
2 
4 
2 
1 
5 
3 
1 
1 
4 
25 

537 
480 

200 
74 
90 
50 
49 
66 
33 
22 
23 
38 
60 

412 

130 
90 
72 

180 

10 
7 

10 
8 

12 
6 

19 

10 
4 
7 
9 

61 
3 
6 

29 

50 

760 
893 
873 
904 
900 
888 
908 
944 
956 
934 
877 
160 
832 
849 
54 
55 

8 

10 

15 

9 

18 

12 

8 

7 

6 

8 

18 

123 

31 

50 

66 

95 

100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 

10 
14 
20 
16 

11 

4 
16 
21 
10 

8 
12 
12 

222 
343 

1030 

529 

Carrot  (vellow) 

900 

Cauliflower 

200 

( 'abbage 

258 

Beans 

244 

Spinach 

106 

Lettuce 

157 

Cucumbers 

230 

Radishes 

317 

Edible  mushrooms  (average).  .  . 
Same  dried  in  the  air  (average).. 
Apples  and  pears 

188 

188 

3250 

Almonds 

1800 
30 

Cocoa 

129 

TABLE  II.— MALT  LIQUORS. 


1000  Parts  by  Weight  contain 


6 

■a 

u 

a  x 

Z    Z 

o 

2 

TJ 

0 

o 

< 

M 

W 

•0 

O 

s 

871 

2 

54 

76 

7 

887 

28 



15 

885 

32 

— 

7 

911 

2 

35 

55 

8 

903 

2 

40 

58 

4 

881 

2 

47 

72 

6 

916 

3 

25 

59 

5 

945 

— 

22 

— 

7 

6 

c 

B 

B 

-3 

6 

3 

>. 

< 

'■3 

3.0 

— 

2.0 

2 

1.5 

2 

1.7 

— 

4.0 

— 

— 

— 

Porter 

Beer  (Swedish) 

"      (Swedish  export). 

Draught-beer 

Lager-beer 

Bock-beer 

Weiss-beer 

Swedish  "Svagdricka" 


13 


ID 

7 

13 


65 
73 


31 

47 


23 


4 

5 
3 

2 
2 
3 

2 

3 


670 


FOOD   TABLES. 


TABLE  III.— WINES  AND  OTHER  ALCOHOLIC  LIQUORS. 


1000  Parts  by  Weight  contain 

u 
o 

o 

o  t-< 

1 

3 

M 

9 

T3.2g 

6 

.9 

o 

>> 

o 

< 

Q  °   • 

^  a 

r-      -  V 
O 

Bordeaux  wine 

883 
863 
776 
801 
808 
795 
774 
791 
790 
479 

94 
115 

90 

94 
120 
170 
164 
156 
164 
263 
460 
550 
442-590 

23 
23 
134 
105 
72 
35 
62 
53 
46 

6 

4 
115 
87 
51 
15 
40 
33 
35 
332 

260-475 

5.9 
5.0 
6.0 
6.0 
7.0 
5.0 
4.0 
5.0 
5.0 

1.0 
1.0 
9.0 
6.0 
2.0 
3.0 
4.0 

2.0 
2.0 
1.0 
2.0 
3.0 
5.0 
3.0 
3.0 
4.0 

Champagne 

Tokay 

[ 60-70 

Sherry 

Madeira 

Swedish  punch 

Brandy 

French  cognac 

Liqueurs 

;. 


in 


INDEX   TO  SPECTRUM  PLATE.  C71 


SPECTRUM  PLATE. 

1.  Absorption  spectrum  of  a  solution  of  oxyhemoglobin. 

2.  Absorption  spectrum  of  a  solution  of  hcemoglobin,  obtained  by  the  action  of  an 

ammoniacal  ferro- tartrate  solution  on  an  oxyhemoglobin  solution. 

3.  Absorption  spectrum  of  a  faintly  alkaline  solution  of  methcemoglobin. 

4.  Absorption  spectrum  of  a  solution  of  hcematin  in  ether  containing  oxalic  acid. 

5.  Absorption  spectrum  of  an  alkaline  solution  of  hcematin. 

6.  Absorption  spectrum  of  an  alkaline  solution  of  hcemochromogen,  obtained  by  the 

action  of  an  ammoniacal  ferro- tartrate  solution  on  an  alkaline-hrematin  solution. 
7    Absorption  spectrum  of  an  acid  solution  of  urobilin. 

8.  Absorption  spectrum  of  an  alkaline  solution  of  urobilin  after  the  addition  of  a  zinc- 

chloride  solution. 

9.  Absorption  spectrum  of  a  solution  of  lutein  (ethereal  extract  of  the  egg-yolk). 


INDEX. 


Absorption,  344 — 357 

,  action    of    putrefactive    pro- 
cesses in  the  intestine  on, 
338 
Absorption  ratio,  183 

of  the  blood  pigments, 
183 
Acceptor,  6 
Accipenserin,  47 

Aittanilid,  behavior  in  animal  body,  542 
Acetha*min,  178 
Acetic  acid  in  intestinal  contents,  333 

in  gastric  contents,  313.  316 
,  passage  of,  into  urine,  520,  539 
Aceto-acetic  acid,  576 

in  urine,  534,  573 
Acetone,  575 

in  urine,  573 
Acetonuria,  573 

Acetophenone,  behavior  in  body,  545 
Act'tvlene,   compound  with  haemoglobin, 

174 
Acetyldichitosamine,  589 
Acetyl  equivalent,  114 
Acetyl  acid  equivalent,  114 
Acetyl-amino  benzoic  acid,  544 
Acetylparaminophenol,  542 
Achilles  tendon,  composition  of,  359 
Acholia,  pigmentary,  277 
Achromatin.  124 
Achroo-dextrin,  105 
Acid  albuminates,  26 

,  properties,  35,  36 
,  formation  in  peptic  di- 
gestion, 303 
,  absorption  of,  345 
Acid  amines,  behavior  in  the  animal  body, 

539 
Acid  equivalent,  113 
Acid  fermentation  of  urine,  581 
Acid  haemoglobin,  172 
Acid  rigor.  393 
Acids,    organic,    behavior   in   the   animal 

l.odv,  464.  .534.  530 
Acidity  of  urine.  463,  464 

of  the  gastric  contents.  314 
of  the  muscles,  376,  393 
Acrite,  92 


Acrolein,  109 

Acrolein  test,  109,  112 

Acroses,  92 

Acrylic  acid  diureid.     See  Uric  acid. 

Actiniochrom,  593 

Adamkiewicz-Hopkin's  reaction,  31,  82 

Adelomorphic  cells,  295 

Adenine,  131,231 

,  properties,   reaction,   and  occur- 
rence, 135 
in  urine,  494 
Adhesion,   importance  in  blood  coagula- 
tion, 190,  191 
Adipocere,  372 
Adrenalin,  237 

-  relation  to  glycosuria,  256 
^•Egagropila,  344 
JErotonometric  method,  610 
Age,  influence  on  metabolism,  654 
Agglutins,  17 

Alanin,  21,  24,  41,  58,  61,  67 
Albumins,  26 

,  general  properties.  33 
,  conversion  into  globulin,  34 
See  also  the  various  albumins. 
Albumin,  detection  of,  in  urine,  550,  552 

,  quantitative       estimation      in 
urine,  552 
See  Proteids. 
Albuminates,  26 

,  properties     and     reactions, 

35—36 
,  ferruginous    albuminate    in 
the  spleen,  232 
Albuminoids,  26,  57 

in  cartilage,  59,  360,  363 
in  the  lens  fibres,  418 
Albumoids,  26,  57 

in  tracheal  cartilage,  58,  360, 

363 
in  lens  fibres,  418 
Albuminose,  in  spermatozoa,  421 
Albuminous  bodies.     See  Proteids. 
Albumoses.      See  Proteoses. 
Alcapton  and  alcaptonuria,  506,  511 — 513 
Alcohol.     See  Ethyl  alcohol. 
Alcohols,  behavior  in  animal  bodv,  540 
Alcoholic  fermentation,  9,  11,  88,  03 

673 


C74 


INDEX. 


Alcoholic  fermentation  in  intestine,  334 

by  tissue  enzymes,  9 
Aldehydases  of  the  liver,  8 
Aldehydes,  behavior  in  the  animal  body, 

540 
Aleuron  grains,  426 
Alexin  es,  156 
Aldoses,  84 

Alimentary  glycosuria,  255,  350 
Alizarin  in  the  urine,  547 
Alkali  albuminates,  26,  29 

,  properties    and    reac- 
tions, 35—36 
,  occurrence      in      the 

brain,  406 
,  occurrence  in  smooth 

muscles,  404 
,  absorption  of,  345 
,  Lieberkiihn's  alkali  al- 
buminate, 35 
Alkali  albumose,  37 

Alkali    carbonates,    physiological    impor- 

'  tance,  636 

,  importance  for  gaseous 

exchange,  600 — 604 

,  action  on  secretion  of 

gastric  juice,  297 
,  action  on  secretion  of 
pancreatic  juice,  322 
.     See    various    tissues 
and  fluids. 
Alkalies,  relation    to    gaseous    exchange, 
187,  188 
,  diffusible   and   non-diffusible   in 

blood.  188 
,  division  of,  in  blood  corpuscles 

and  plasma,  188,  201 
.     See  also  the  various  fluids  and 
tissues. 
Alkali  phosphates  in  urine,  463,  491,  529 
,  occurrence.        See  the 
various    fluids    and 
organs. 
Alkali  proteose,  37 
Alkali  urates,  462,  491 

in  calculi,  582 
in  sediments,  462,  491,  582 
Alkali  earths,  elimination    by    the    intes- 
tine, 529,  535 
in  urine,  535 
in  bones,  365,  366 
,  insufficient  supply  of,  368, 
636 
Akaline  fermentation  of  urine,  581 
Alkalinity,  determination  of,  in  blood,  160 
Alkaloids,  action  on  muscles,  393 

,  passage  of,  into  urine,  547 
,  retention  by  the  liver,  239 
Alkvl  sulphide  of  the  skunk,  594 
Allantoic  fluid,  435 

Allantoin,  properties  and  occurrence,  499, 
500 
in  transudates,  219,  435 
,  formation  from  uric  acid,  489, 
499 


Alloxan,  25,  484 

Alloxuric  bases,  130,  494,  495,  496 

Alloxuric  bodies,  130 

Alloxyproteic  acid,  524,  525 

Almen-Bottger-Nylander's  sugar  test,  94, 

562 
Ambergris,  344 
Ambrain,  344 
Amid  nitrogen,  18,  19 
Amino  acids,  relation  to  formation  of  uric 
acid,  488 
,  relation  to  formation  of  urea, 

469,  539 
,  formation    in    putrefaction, 

21,  334 
,  formation  from  protein  sub- 
stances,   21,   24,    66—82, 
334 
,  formation  in  tryptic  diges- 
tion, 329 
,  separation  and  preparation, 

73 
,  behavior  in  the  animal  body, 
539 
Amino-acetic  acid.     See  Glycocoll. 
Amino-benzoic  acids,  behavior  in  the  ani- 
mal body,  544 
Amino-caproic  acid.     See  Leucin. 
Amino-cerebrinic  acid  chloride,  412 
Amino-cerebrinic  acid  glucoside,  411 
Amino-cinnamic  acid,  542 
Amino-glutaric  acid,  24,  71 
Amino-ethyl  sulphonic  acid.     See  Taurin. 
Amino-phenyl-acetic  acid,  behavior  in  ani- 
mal body,  543 
Amino-phenyl-propionic  acid,  formation  in 

the  putrefaction  of  proteids,  22,  501 
Amino-phenyl-propionic  acid,  behavior  in 

the  animal  body,  543 
Amino-propionic  acid,  67 
Amino-pyrotartaric    acid.     See   Glutamic 

acid. 
Amino  sugar,  23,  51 

Amino-succinic  acid.     See  Aspartic  acid. 
Amino-thiolactic  acid,  behavior  in  the  ani- 
mal bodv,  540 
Amino-valerianic  acid,  21, 24, 41,  58,  60,  68 
Amidulin,  104,  290 

Ammonia,  formation  in  proteid  putrefac- 
tion, 334 
,  formation    from    protein    sub- 
stances, 20,  25,  329,  334 
,  formation  in  tryptic  digestion, 

329 
,  occurrence  in  blood,  202,  470, 

534 
,  occurrence  in   urine,  464,  468, 

533 
,  elimination    after    administra- 
tion of  mineral  acids,  464, 534 
,  elimination  in  disease,  534 
,  elimination  in  diseases  of  the 

liver,  468 
,  after  extirpation  or  atrophy  of 
the  liver,  472 


INDEX. 


675 


Ammonia,  estimation  of,  in  urine,  535 
Ammonium  salts,  relation  to  formation  of 
glycogen,  248 
,  relation  to  formation  of 

urea,  470 — 172 
,  relation  to  formation  of 
uric  acid,  487 
Ammonium-magnesium  phosphate  in  uri- 
nary calculi,  585 
Ammonium-magneeium  phosphate  in  In- 
testinal calculi,  343 
Ammonium-magnesium  phosphate  in  uri- 
nary sediment,  583 
Ammonium  sulphate,  method  of  separat- 
ing proteoses,  38, 
K),  43 
,  method  of  separat- 
i  n  g        carbohy- 
drates, 106,  245 
Ammonium  urate  in  urinary  sediments, 
584,  585 
in  urinary  calculi,  582 
Amniotic  fluid,  435 
Amphicreatine,  386 
Amphopeptone,  39 
Amygdalin,  14 
Amylodextrin,  105 
Amyloid,  26,  54,  361 

,  vegetable,  107 
Amyloid  degeneration,  bile  in,  277 

,  chondroitin  -  sul- 
phuric   acid    in 
the  liver  in,  361 
Amylolytic  enzymes,  13,  318,  324 
Amvlopsin,  324 
Amylum.     See  Starch. 
Anemia,  pernicious,  208 
Aniline,  behavior  in  the  animal  body,  542 
Anisotropous  substance,  376 
Antedonin,  593 
Antialbumate,  303 
Antialbumid,  303 
Antialbumose,  38 
Antienzymes,  17,  155,  299,  318 
Antifebrin,  relation  to  elimination  of  uro- 
bilin, 517 
Antimony,  passage  of,  into  milk,  459 

,  action  on  the  elimination  of 
nitrogen,  468 
Antipeptone,  39,  41,  44,  45 
Antipyrin,  relation  to  formation  of  gly- 
cogen, 248 
,  action  on  the  urine,  546 
Antitoxins,  17 
Anuria,  in  cholera,  596 
Aorta  elastin,  59 
Apatite  in  bone-earths,  366 
Arabinose,  85,  87,  88,  90 

,  relation  to  formation  of  gly- 
cogen, 247 
Arabinosimine,  86 
Arabite,  85 

Arachidic  acid,  108,  440 
Arachnoidal  fluid,  218 
Arbacin,  49 


Arbutin,  relation  to  formation  of  glyco- 
gen, 247 
,  behavior  in  the  animal  body,  606 
Arginin,  18,21.24,43,45,47,48,68,82,  77 
Argon  in  blood,  598 
Arnold's  aceto-acetic  acid  reaction,  ">77 
Aromatic  compounds,  behavior  in  animal 

body,  541—547 
Arsenic,  in  the  animal  body,  157,  459,  588, 
596 
action    on    the    elimination    of 
nitrogen,   168 
Arsenious  acid,  action  on  peptic  digestion, 

303 
Arseniuretted  hydrogen,  poisoning  with, 

279,  281,  555 
Arterin,  165 

Ascitic  fluids,  51,  219,  221 
Asparagin,  70 

,  relation   to  synthesis  of  pro- 

teidfl,  25 
,  relation  to  formation  of  gly- 
cogen, 248 
,  nutritive  value,  645 
Asparaginic  acid.     See  Aspartic  acid. 
Aspartic  acid,  70 

,  relation    to    formation    of 

uric  acid,  488 
,  relation     to  formation     of 

urea,  469 
,  formation  from  proteid,  21, 

24,  41,  58,  60,  61 
,  behavior  in  the  organism, 
469,  488,  540 
Asparagus,  odoriferous  bodies  of,  in  the 

urine,  547 
Assimilation  limit,  350 
Ass's  milk,  449,  450 
Atmidalbumin,  40 
Atmidalbumose,  40 
Atmidkeratin,  58 
Atmidkeratose,  58 

Atropine,  action    of,    elimination   of   uric 
acid,  486 
,  on  the  secretion  of  saliva,  293 
Auto-digestion.     See  Autolysis. 
Auto-intoxication,  17 
Autolysis,  11 

,  substances   retarding   coagula- 
tion produced  in,  197 
.     See  also  the  various  organs 
and  tissues. 
Auto-oxidation,  3,  5,  6 

Bacterial  proteids,  19 

Bacteria  urese,  581 

Banting  cure,  666 

Beer-vineger  bacteria,  enzyme  of,  11 

Beeswax,  115 

Bi'la's  acetone  reaction,  576 

Bence-Jones  proteid,  552 

Benzaldehyde,  oxidation  of,  5 

,  substituted  aldehyde,  be- 
havior in  the  animal 
body,  544 


676 


INDEX. 


Benzoic  acid,  formation  from  protein  sub- 
stances, 23,  500 
,  passage  of,  into  sweat,  596 
,  behavior  in  the  organism,  2, 

500,  543 
,  occurrence  in  the  urine,  503 
,  substituted    benzoic    acids, 
action  in  body,  544 
Benzene,  23,  60 

,  behavior   in   the   animal   body, 
541,  542 
Benzoyl-amino-acetic  acid.     See  Hippuric 

acid. 
Benzoyl-cystin,  75 
Benzoar-stones,  344 
Bial's  reagent,  572 
Bifurcated  air,  609 
Bile,  259—282 

,  analysis  of,  275,  276 

,  antiseptic  action,  338,  339 

,  enzymes  of,  274 

in  disease,  277 
,  influence  on  proteid  digestion,  332 
,  on  the  emulsification  of  fats,  332, 333, 

354 
,  on  the  secretion  of  bile,  261 
,  on  the  absorption  of  fat,  332,  352 — 

354 
,  on  tryptic  digestion,  328,  333 
,  molecular  concentration,  275 
,  passage  of  foreign  bodies,  277 
,  occurrence  of,  in  urine,  558,  559 
,  occurrence   of,   in  gastric  contents, 
313,  332 
in  meconium,  342 
,  decomposition  in  the  intestine,  337 
,  formation  of,  278—282 
Bile-concretions,  282 
Bile-pigments,  269—274 

,  origin  and  formation,  278 

—282 
,  reactions,    271,    272,    273, 

559 
,  passage  of,  into  urine  559 
Biliary  fistula?,  259 

,  influence  of,  on  intestinal 
putrefaction,  239 
Biliary  fistula?,  influence  on  the  want  of 

food,  339 
Bile-salts,  262 
Bile-acids,  262—269 
in  pus,  228 
in  urine,  356,  558 
,  detection  of,  269 
,  absorption  of,  356 
,  origin  of,  278,  279 
Bile-mucus,  261 
Bilianic  acid,  266 
Bilicvanin,  269,  272,  273 
I'.ilifulvin,  269 
Bilifuscin,  269,  273 
Bilihumin,  269,  273 
Bfliphsrin,  269 
Biliprasfa,  269,  273 
Bilipurpurin,  273 


Bilirubin,  269 

,  relationship  to  blood-pigments, 

177,  270,  280 
,  relationship  to  haematoidin,  179, 

279,  280 
,  putrefaction  of,  337 
,  occurrence,  269 
Biliverdin,  272 

in  faeces,  342 
Biogen  molecule,  6 
Bioses,  16 

Bismuth,  passage  of,  into  milk,  469 
Birotation,  88 
Bitch's  milk,  450,  455 
Biuret,  25,  473 
Biuret  base,  cleavage  of,  330 
Biuret  reaction,  25,  31,  46,  473 
Blister  fluid,  224 
Blonds,  milk  of,  454 
Blood,  142—210 

,  general  behavior,  142,  187 — 192 
,  analyses,  quantitative,  198 — 203 
,  analyses,  physico-chemical,  160 
,  arterial  and  venous,  165,  203,  599 
,  defibrinated,  143 
,  asphyxiation,  165,  599 
,  quantity  of,  in  the  body,  209 
,  detection,  chemico-legal,  181 
,  behavior  in  starvation,  206 
,  composition  under  various  condi- 
tions, 200—209 
in  gastric  contents,  313 
in  urine,  554 — 557 
Blood-casts,  555 
Blood-clot,  143,  189 

Blood  coagulation,  142, 143, 146, 189—198 
Blood-corpuscles,  white,  1S5,  186 

,  number  of,  185,  208 
,  relation  to  coagulation, 

185,  191 
,  red,  161—164 
,  number  of,  161,  207,  208 
,  relation    to    high    alti- 
tudes, 207 
,  passage  of,  into  urine, 

554 
,  permeability,  164 
,  composition,  184,  185 
Blood  gases,  598—604 
Blood-pigments,  164 — 184 
in  bile,  277 
in  urine,  554 — 556 
Blood-plasma,  144 — 153 

,  composition  of,  157,  201 
Blood-plates,  185,  186,  195 

,  relation    to    coagulation   of 
blood,  191 
Blood-serum,  143,  153—161 

,  anti-enzymes  of,  155 
,  enzymes  of,  155 
,  precipitins  of,  156 
,  composition  of,  157 
Blood-spots,  1*1 
Blond  transfusion,  210 
Blue  stentorin,  593 


INDEX. 


677 


Boar  spermatozoa,  421 
Bom  reaction  f<>r  IK'l,  314 

Bones  and  bone  tissues,  364 — 370 

in  starvation,  530, 
631 
Bone-earths,  365 
Bone  marrow,  367 

Bonellin,  593 
Borneol,  522 

I'm. ttclicr's  spermin  crystals,  420 
Bottger-Almen's  sugar  test,  94,  562 

Bowman's  disks,  377 

Brain,   10(1-414 

Bromadenine,  132 

Bromanil,  25 

Bromhypoxanthine,  132 

Bromides,  behavior  to  secretion  of  gastric 

juice,  307 
Bromine,  passage  of,  into  saliva,  293 
Bromoform,  from  proteid,  23 

,  behavior  in  the  animal  body, 
540 
Bromtoluene,  behavior  in  the  animal  body, 

5 1 1 
Brunettes,  milk  of,  454 
Brunner's  glands,  316 
Buccal  mucus,  288 
Buffv  coat,  189 
Bufidin,  594 
Bufotalin,  594 
Bufotenin,  594 
Bufotin,  594 
Bull,  spermatozoa,  421 
Bursa'  mucosa',  contents  of,  226 
Butalanin,  65 
Butter-fat,  440 

,  absorption  of,  354 
Butterflv,  pigment  of  wings,  485,  593 
Buttermilk,  4  19 
Butyl    alcohol,    behavior   in   the   animal 

body,  .",11 
Butyric  acid,  21 

in  urine,  520 
in  gastric  contents,  298 
in  milk  fat,  440 
Butyric-acid  fermentation,  4,  88,  438 

in      intestine, 
336 
Butylmercaptan,  594 
Butyrinase,  in  blood,  155 
Byssus,  26,  65 

Cadaverin,  16 

in  intestine,  580 
in  urine,  526,  580 
Caffeine,  131 

,  behavior   in    the   animal   body, 

494 
,  action  on  the  muscles,  393 
Calcium,  lack  of,  in  food,  368,  636 

,  occurrence.      See  various  tissues 
and  fluids. 
Calcium  carbonate  in  urine,  583 

in      urinary      calculi, 
585 


Calcium  carbonate  in  urinary  sediments, 
583 
in  bones,  366,  368 
in  tartar,  L")  1 
Calcium  casein,  1 II 
Calcium  oxalate  in  urine,  498 

in  urinary  sediments,  582 
in    urinary    calculi,    584, 
585 
Calcium  phosphate,  relation  to  the  coagu- 
lation of  fibrinogen, 
192 
,  relation  to  the  coag- 
ulation   of    casein, 
441 
,  occurrence  in  the  in- 
testinal        concre- 
tions, 343 
in  the  urine,  463,  529, 

530,  535 
in  urinary  sediments, 

583 
in  urinary  calculi,  585 
Calcium  salts,  elimination,  529,  535 

,  importance  to  coagulation 
of  the  blood,    143,    147, 
193 
,  importance  to  coagulation 

of  milk,  442 
.     See  various  calcium  salts. 
Calcium  sulphate    in    urinary    sediments, 
583 
,  physiological       action, 
140 
Calculi,  salivary,  294 
,  intestinal,  343 
,  urinary,  584 
Calories  of  foodstuffs,  624 — 628 

of  different  rations,  660 — 665 
Campho-glucuronic  acid,  100,  522,  546 
Camphor,  behavior  in  the  animal  body, 

522, 546 
Cane-sugar.     See  Saccharose. 
C'apranica's  reaction  for  guanine,  134 
Capric  acid,  108,  440,  451 
Caproic  acid,  108,  440,  451 
Caprylic  acid,  108,  440 
Caramel,  93,  101 
Carbamic  acid,  480 

in  blood,  156,  470 
in  urine,  470.  471,  472,  4S0 
,  poisonous  action,  470 
Carbamic-acid  ethvloster.   isn 
Carbazol,  behavior  in  body,  542 
Carbothoxyldiclvcylleucin  ester,  25 
Carbethoxyltrisflyevlglycin  ester,  25 
Carboglobulinic  acid,  602 
Carbohirmoglobin,  174 
Carbohydrates,  83—108 

,  importance  in  fat  forma- 
tion. 374,  650 
,  importance     in     glveogen 

formation.  249.  250 
,  importance    for    muscular 
activity,  395,  400,  401 


678 


INDEX. 


Carbohydrates,  action  on  proteid  metabo- 
lism, 638,  645—650 
,  action  on  intestinal  putre- 
faction, 338,  340,  504 
,  absorption  of,  349 — 353 
,  inadequate  supply  of,  638 
.     See  also  the  various  car- 
bohydrates. 
Carbolic  acid,  action  on  peptic   digestion, 
303] 
.     See  also  Phenol. 
Carbolic  urine,  506 

Carbon,  relation  to  nitrogen  in  the  urine, 
537,  538 
,  calorific  value,  627 
Carbon  dioxide  in    the    blood,    598 — 603, 
608—612 
in  the  blood  in  diabetes, 

602 
in  the  blood  in  poisoning 
with  mineral  acids,  602 
in  the  intestine,  334 — 336 
in  the  lymph,  212,  603 
in  the  stomach,  308 
in  the  muscles  during  rest 

and  activity,  395,  399 
in    the    muscles    in    rigor 

mortis,  394 
in  the  secretions,  603 
in  transudations,  604 
action  on  the  secretion  of 

gastric  juice,  296 
elimination,  dependence  of 
external       temperature 
upon, 658 
elimination  in  rest  and  ac- 
tivity, 395, 399, 656,  657 
elimination  by  the  skin, 597 
elimination      in      various 
ages,  652,  653 
Carbon-dioxide  haemoglobin,  174,  600 
Carbon-monoxide  poisoning,  173, 256, 389, 
468 
action  on  the  formation  of  lactic  acid, 

389 
action  on  the  elimination  of  nitrogen, 

468 
action  on  the  elimination  of  sugar, 
256,  389 
Carbon-monoxide  haemoglobin,  172,  173, 

183 
Carbon-monoxide  methsomoglobin,  174 
Carbon-monoxide  blood  test,  Hoppe-Sey- 

ler's,  173 
f  arminic  acid,  593 
C.'irnic  acid,  385 
Carniferrin,  385 
famine,  131,  385 

in  urine,  494 
Parnosin,  383,  385 
Carp,  sperma  of,  47,  48 

eggs  of,  55 
Cartilage,  54,  360—364 

,  quantity  of  ash,  364 

,  behavior  to  gastric  juice,  304 


Cartilage,  behavior  to  pancreatic  juice,  330 
Cartilage  gelatine,  363 
Caseid,  441 

Casein,  26,  80,  440,  451 
,  origin  of,  457 
,  from  woman's  milk,  451 
,  from  cow's  milk,  440 
,  quantitative  estimation  of,  445 
,  absorption  of,  345 
,  behavior  towards  rennin,  305,  441 , 

442 
,  behavior  to  gastric  juice,  309,  442, 
451 
Casein,  heat  of  combustion,  625 
Caseinogen,  442 
Caseoses,  39 

,  relation  to  the  coagulation  of  the 
blood,  143 
Castor  bean,  17 
Castoreum,  594 
Castorin,  594 
Catalases,  6,  7,  8,  444 
Catalysators,  8,  15 
Cathsemoglobin,  174 
Catheterization  of  the  lungs,  608 
Cat's  milk,  450 
Cell,  animal,  116—142 
Cell  constituents,  primary  and  secondary, 

117 
Cell  fibrinogen,  231 
Cell  globulins,  117,  118,  163 
Cell  membrane,  119,  304 
Cell  nucleus,  124 

,  relation    to    coagulation    of 

fibrinogen,  186,  191 
,  to  pepsin  digestion,  304 
Cellulose,  106,  107 

,  fermentation  of,  107,  332,  337 
Cement,  369 
Cephalin,  411 
Cephalic  acid,  411 
Cerebrin,  227,  407,  408 

,  properties  and  behavior,  409, 410 
in  pus,  227 
Cerebrinin  phosphoric  acid,  412 
Cerebrinic  acid,  412 
Cerebron,  407,  411 
Cerebrosides,  408,  409,  411 
Cerebrospinal  fluid,  223,  413 
Cerolein,  115 
Cerotic  acid,  115 
Cerumen,  594 
Cetin,  115 
Cetyl  alcohol,  115 
Chalaza  429 

Charcot's  crystals,  209,  420,  615 
Cheno-taurocholic  acid,  265 
Children's  urine,  462,  468,  499 
Chitaminic  acid,  98 
Chitaric  acid,  99 
Chitin,  65.  98,  589 

,  behavior  in  tryptic  digestion,  330 
Chitosamine.     See  Glucosamine. 
Chitosan,  51,  590 
Chitose,  99 


INDEX. 


679 


Chloral  hydrate,  behavior  in  the  animal 

body,  522,  541 
Chlorates,  poisoning  with,  171,  555 
( Ihlorazol,  23 
Chlorbenzene,    behavior    in    the    animal 

body,  S 16 
Chlorides,  elimination  by  the  urine,  626 
.  elimination  by  the  sweat,  596 
,  action  <>n  proteid  metabolism, 

651 
,  insufficient  supply  of,  635 
.    Bee  also   various   fluids   and 
tissues. 
Chlorochrome,  241 
( Jhlorocruorin,  is  I 

Chloroform,  action  on  the  elimination  of 
chlorides,  527 
,  action  on  the  muscles,  393 
,  act  ion  upon  proteids,  29 
,  behavior  in  the  animal  body, 
540 
Chlorophan,   U6 
Chlorophyll,  2,  593 

,  relation    to    blood-pigments, 
165,  180 
Chlorosis,  208 
Chlorphenylcystein,  546 
Chlorphenylmercapturic  acid,  546 
Chlorrhodinic  acid,  228 
Chlortoluene,  behavior  in  the  animal  bodv, 

oil 

Chola^ogues,  261 
( Iholanic  acid,  267 
Cholecyanin,  270,  272,  516 
Choleic  acid,  2i>7 
Cholepyrrhin,  269 
Cholera,  blood  in,  202 
,  sweat  in,  596 
,  ptomaines  in,  16 
Cholera  bacilli,  behavior  with  gastric  juice, 

312 
Cholesterilene,  282 
Cholesterilin,  282 
Cholesterin,  2S2 

,  preparation,  285 
in  blood-serum,  154 
in  sputum,  615 
in  the  bile,  262,  274,  277 
in  .^all-stones,  282 
in  the  brain,  407,  413 
in  the  urine,  579 
in  urinary  calculi,  586 
,  importance  in  the  life  proc- 
esses of  the  cell,  117,  124 
Cholesterin  calculi,  282 
Cholesterin  ester  in  blood-serum,  154 
Cholesterin  fat,  as  protective  fat,  593 
Cholesterin-propionic  ester,  283 
Cholesterinic  acid,  2(>i> 
Cholesteron,  282 
Choletelin,  269,  272 

,  relation  to  urobilin,  516 
Cholic  acids,  262,  266 

,  preparation,    267.     See    also 
the  individual  cholic  acids 


Choline,  1  •  i .  120,  274,  413 
<  Iholohsmatm,  -7.i 
( Sholoidic  acid,  260 
( Iholylio  acid,  266 
Chondrigen,  61,  360 
Chondrin,  til 

in  pus,  228 
Chondrin  balls,  363 
( ihondroalbumoid,  365 
Chondroitie  acid,  360 
( Shondroitin,  361 

(hondroitin-sulphuric     acid,  30,    51,    51, 

360,  361 
in  urine,  523, 

554 
in     kidneys, 
162 
(  hondromucoid,  51,  54,  360,  365 
Chondroproteids,  51,  54 

in  the  urine,  553 
Chondrosin   from     chondroit  in-sulphuric 
acid,  361 
from  sponges,  54 
Chorda  saliva,  287 
Choroid  coat,  418 

pigment  of,  591 
Chromatin,  124 
Chromhidrosis,  596 
Chromogens  in  urine,  514 

in  suprarenal  capsule,  237 
Chrvsophanic  acid,  action  on  urine,  547 
Chyle,  211—214 
( 'hvluria,  579 
Chyme,  308 

investigation  of,  313 — 316 
Chymosin,  13,  17,  155,  304,  442 

detection  in  gastric  contents, 

313 
in  urine,  534 

occurrence  in  the  pancreas,  330 
.     See  also  llennin. 
Cilianic  acid,  266 
Citric  acid  in  milk,  440,  448,  453 
Clupein,  17,  18 
Coagulated  proteids,  2i>,  17 
Coagulation  of  the  blood,  142,  143,  1  17, 
1*9—197 
,  intravascular,  196 
of  milk.  138,   Ml.  451 
of  muscle-plasma,  o77.  3S1, 
393 
Coagu loses.  44 
(  occinic  acid,  593 
( loccygeal  glands,  594 
Cochineal,  593 
Cochinfllic  acid.  593 
Codfish,  spermatozoa,  49 
Coefficient.  Haser's,  537 

,  respiratorv.  375,  399,  622,  655, 

656 
,  dissociation,  159 
,  extinction,  182,  183 
,  urotoxic,  526 
Coffee,  action  on  metabolism,  652 
Collagen,  26,  61—64,  358,  360,  303 


680 


INDEX. 


Colloid,  53,  235,  422 
Colloid  corpuscles,  423 
Colloid  cysts,  422 
Colon,  exclusion  of,  357 
Coloring-matters.     See  various  pigments. 
Colostrum  of  woman's  milk,  454 

of  cow's  milk,  449 
Colostrum  corpuscles,  449 
Comma    bacillus,    behavior   with    gastric 

juice,  312 
Compound  proteids,  26,  51 — 57 

.     See  also  the  different  groups 
of  protein  substances. 
Conalbumin,  430 
Conchiolin,  26,  65,  66 
Concentration,    molecular.       See   various 

fluids. 
Concrements.     See  various  calculi. 
Cones  of  the  retina,  pigment  of,  416 
Conglutin,  calorific  value  of,  625 
Connective  tissues,  59,  358,  359 
Copaiva  balsam,  action  on  the  urine,  547 
Copper  in  blood,  156,  201 
in  bile,  262 
in  biliary  calculi,  282 
in  ha?mocyanin,  184 
in  protein  substances,  18 
in  turacin,  592 
Cornea,  364,  418 
Cornein,  26,  65 
Cornicrystallin,  65 
Corpora  lutea,  422 
Corpulence,  diet  cures  for,  665,  666 
Corpuscula  amylacea,  412 
Cow's  milk,  437—448 

,  general    behavior,    437,    438, 

439 
,  analysis  of,  446 — 448 
,  anti-putrefactive    action    of, 

338,  504 
,  coagulation  with  rennin,  305, 

313,  439 
,  behavior  in  the  stomach,  451 
,  composition  of,  448 
Cream,  449 

Creatine,  relation   to   formation   of   urea, 
•     383,  469 
,  relation    to    muscular    activity, 

397,  400 
,  properties  and  occurrence,  383, 
384 
Creatinine,  relation  to  muscular  activity, 
397,  400,  481 
,  properties  and  occurrence,  481, 
482 
zinc  chloride,  482 
Cresol,  21,  334,  504,  505 
(  Yesol-sulphuric  acid,  504,  505 
Cririosin,  412 
Crotonic  acid,  578 
Cruor,  143 
Crasocreatinine,  386 
(  i  M-taceorubin,  593 

I  Jrusta  inflammatoria  or  phlogistica,  189 
Crystalbumin,  417 


Crystalfibrin,  417 

Crystallins,  417 

Crystalline  lens,  416—418 

Crystalline  seralbumin,  134 

Cumic  acid,  543 

Cuminuric  acid,  543 

Curd,  442 

Cyanhydrines,  85 

Cyanmethsemoglobin,  172 

Cyanocrystallin,  433,  592 

Cyanuric  acid,  473,  484 

Cyanurin,  514 

Cyclopterin,  47,  48 

Cymene,  543 

Cyprinine,  48 

Cystein,  20,  21,24,  76 

,  conjugation  in  animal  body,  546 
,  behavior    in    the    animal    body, 
540 

Cysteinic  acid,  75 

Cystin,  19,  21,  24,  58,  75,  76,  279,  580 
,  occurrence  in  urine,  523,  580 
,  occurrence  in  urinary  sediments, 

583 
,  occurrence  in  urinary  calculi,  586 
,  occurrence  in  sweat,  596 
,  synthesis,  76 
,  behavior  in  the  animal  body,  279 

Cystinuria,  16,  75,  526,  580 

Cystome,  poliferous,  422 

Cysts,  tapeworm,  225 
,  ovarial,  422 — 426 
,  thyroid,  235 
,  mucoid  substances  of,  51 

Cytin,  231 

Cytoglobin,  26,  118,  192,  231 

Cytosin,  127,  130,  138,  139 

Cytotoxins,  156 

Cytozym,  195 

Damaluric  acid,  526 
Damolic  acid,  526 
Defibrinated  blood,  143 
Dehydrocholic  acid,  266 
Dehydrocholeic  acid,  267 
Delomorphoic  or  parietal  cells,  295 
Denige's  reaction  for  uric  acid,  491 
Denige-Morner's  tyrosin  test,  73 
Dentin,  366,  369 
Dermoid  cyst,  425 
Desaminoalbuminic  acid,  37 
Descemet's  membrane,  53,  364 
Desoxycholic  acid,  266,  267 
Deuteroelastose,  60 
Deuteroproteose,  39,  45,  551 
Deuterogelatose,  63 
Deuteromyosinose,  40 
Deuterovitellose,  40 
Dextrins,  105 

,  formation  from  starch,  105,  290, 
325 

,  loading  the  stomach  with,  307 

,  occurrence  in  the  gastric  con- 
tents, 312 

,  occurrence  in  muscles,  388 


INDEX. 


681 


Dextrine,  occurrence  in  portal  blood,  204, 

350 
Dextrin-like  substance  in  the  urine,  521 
Dextrose,  92  -96 

in  blood,  154,  204,  252,  254 
in  urine,  254,  521,  501—570 
in  the  Lymph,  21 1 
in  muscles,  388 

in  the  vitreous  humor,  416 
,  preparation  of,  95 
,  calorific  value  of,  625 
,  detection,  93,  94,  561—565 
,  reactions  of,  94,  95 
,  absorption  of,  350 
,  quantitative  estimation,   565 — 
570 
Diabetes  mellitus,  254—259,  561 

,  elimination    of   ammo- 
nia by  the  urine  in, 
534 
,  relationship  of  the  liver 

to,  257 
,  relationship  of  the  pan- 
creas to,  257,  258 
,  blood  in,  202,  254 
,  amount    of     sugar    in 

blood  in,  202,  254 
,  urine  in,  469,  537,  551 
,  CO,  in  the  blood  in,  602 
,  oxybutyric  acid  in  the 

blood  in,  603 
,  oxybutyric  acid  in  the 
urine  in,  534,  574,  578 
Diacetic  acid.     See  aceto-acetic  acid. 
Dialuric    acid,   relationship  to   formation 

of  uric  acid,  4ss 
Diamid,  poisoning  with,  409 
Diamino  acids,  18,  24,  74 — 80 
Diamines  in  the  urine,  21,  526,  5^0 

in  the  gastric  contents,  5S0 
Diamino-caproic  acid.     See  Lysin. 
Diamino-valerianic  acid,  18.   See  Ornithin. 
I  >iastatic  enzymes,  13,  155,   252,  290,  324 
See    also    other   en- 
zymes. 
Diastase  in  the  blood,  155 
Diazobenzol-sulphonic  acid,  reaction  with 

sugar,  95 
Dibenzoylornithin,  78 
Dicalcium  casein,  441 
Dichlorpurin,  131 
Diet  cures,  665,  666 
Diet  for  various  classes  of  people,  660,  663, 

664,665 
Digestion,  286—353 
Digestibility  of  foodstuffs,  310,  348,  351, 

353,  354 
Pimethvlketone.     See  Acetone. 
Dioxyacetone,  92 
Dioxybensenes,  506,  542 
Dioxynaphthalene,  542 
Dipalmitylolein,  109 
Dipeptides,  25 

Diphtheria      toxins,     action      upon     the 
gastric  juice,  312 


Disaccharides,  100 

in  urine,  350,  570 
,  inversion  of,  318,  332,  350 

as  glycogen  formers,  250 
Dissociation  decree,  1 .7.1 
Dissociation  coefficient,  159 
Difltearyllecithin,  L20 
Distearylpalmitin,  109 
Doeglic  acid,  112 
Dog's  milk,  450,  455 
Dolphin's  milk,  450 
Donne's  pus  test,  55S 
Dotterplatchen,  27,  426 
Dropsical  fluid,  221 
Dulcite,  85 

,  relationship  to  formation  of  gly- 
cogen, 247 
Dysproteose,  39 
Dyslysins,  268 
Dyspeptone,  303 

Dyspncea,  action  on  proteid  transforma- 
tion, 468,  655 

Ear,  fluids  of,  418 

Earthy  phosphates,  elimination     by     the 
urine,  463,  535 
,  absorption  of,  356 
,  solubility     in     fluids 
rich  in  proteid,  369 
occurrence    in    bone- 
earths,  366—370 
,  occurrence  in  calculi, 

282,  343,  534 
,  occurrence     in     sedi- 
ments, 583 
See   also    different 
earthy  phosphates. 
Ebstein's  diet  cure,  665 
Echinochrom,  1S4 
Echinococcus  cysts,  cyst  wall,  590 

,  cyst  contents,  225 
Eck's  fistula,  471 
Edestair,  49 
Edestin,  23,  34,  49,  80 
Edible  bird's  nests,  53 
Eel,  flesh  of,  404 
Egg,  426 

,  hen's  426 — 436 

,  absorption  in  the  intestine,  34S 
,  incubation  of,  433 — 135 
Etc  albumin  (see  Ovalbumin),  430 
Ksrii-sliell,  57,  272,  432 
Egg  yolk,  428 

Ehrlich's  test  for  bile-pigments,  560 
glucosamine  test,  99 
urine  test,  579 
Eiselt's  reaction  for  melanin,  557 
Elaidic  acid,  112 
Elaidin,  112 
Elastin  proteoses,  60 
Elastin  peptone,  60 
Elastin,  20,  26,  59,  60,  80,  119 

,  behavior  with  gastric  juice,  304, 

358 
,  behavior  with  trypsin,  330 


6S2 


INDEX. 


Elephant  bones,  366 
milk,  450 
tusk,  370 
Ellagic  acid,  444 
Emulsin,  14 
Emydin,  433 
Enamel,  369 
Encephalin,  40S,  410 
Endolymph,  418 
Endosoma,  162 

Energy,  potential,  of  foodstuffs,  624—623 
Enterokinase,  318,  319,  321 
Enzymes  in  general,  10 — 15 

,  amylolytic  or  diastatic,  13,  238, 

252,  274,  288,  324 
,  fat-splitting    or    lipolytic,     13, 

325 
,  coagulating,     13.     See     rennin 

and  fibrin  ferment. 
,  glucoside-splitting,  13,  14 
,  -urea-splitting,  13 
,  oxidizing.     See  Oxidases. 
,  proteolytic,  13,  228,  232,  274 
,  zymogens  of,  11 

See  also  the  various  enzymes, 
tissues,  organs,  and  fluids. 
Epidermis,  57,  538 
Epiguanine,  131,  494,  496 
Epinephrin,  237 
Episarkine,  131,  494,  496 
Erepsin,  318 

,  importance  for  absorption,  347 
Erucic  acid,  108 

,  absorption  of,  354 
Ervthrite,  relation  to  glycogen  formation, 

247 
Erythrocytes,    162,    163,    164.     See    also 

red   blood-corpuscles. 
Erythro-dextrin,  105,  290 
Erythropsin.     See  Visual  purple. 
Es'bach's  estimation  of  proteid  in  urine, 

553 
Ethal,  115 

Ether,  action  on  the  blood,  162,  164 
,  action  upon  proteids,  29 
,  action  on  the  secretion  of   gastric 

.  juice,  296 
,  action  on  the  muscles,  393 
Ethereal  sulphuric  acids  in  the  bile,  262, 

274 
Ethereal  sulphuric  acids  in  the  urine,  503, 

510,  545 
Ethereal  sulphuric  acids,  synthesis  of,  in 

the  liver,  239 
Ethyl  alcohol,  production    in    the    intes- 
tine, 334 
,  passage  of,  into  milk,  461 
,  behavior     in     the     animal 

body,  651 
,  action  on  the  secretion  of 

gastric  juice,  296,  303 
,  action  on  the  muscles,  393 
.  action  on  metabolism,  651 
,  action  on  digestion,  303 
,  action  on  proteids,  29,  30 


Ethylamine,  solvent  for  uric  acid,  491 
Ethyl   benzene,    behavior  in   the   animal 

body,  542 
Ethylene  glycol,  relation  to  formation  of 

glycogen,  24S 
Ethylenimine.     See  Spermin. 
Ethylidene-lactic     acid,     3S8.     See     also 

other  lactic  acids. 
Ethylmercaptan,   behavior  in  the  animal 

body,  540 
Ethyl-sulphuric     acid,    behavior    in    the 

animal  body,  540 
Ethyl  sulphide,   formation  from  proteid, 
20,  21 ,  24 
,  behavior    in  the  animal 
body,  540 
Euglobulin,  149 
Euxanthic  acid,  100 
Euxanthon,  545 
Euxanthonic  acid,  546 
Excrements,  340,  341,  342 

in  dogs  with  biliary  fistula, 

339 
in  starvation,  618 
Excreta,  of  the  animal  organism,  617 — 622 
division  by  the  various  channels, 
618 
Excretin,  342 
Excretolic  acid,  342 
Exostosis,  368 
Expectorations,  614,  615 
Extinction  coefficient,  182,  1S3 
Extracellular  action  of  enzymes,  11 
Exudates,  217—226 
Eye,  414—418 

Faeces.     See  Excrements. 
Fat,  origin  in  the  body,  372—374,  640 
,  general  properties,  detection  and  oc- 
currence, 108 — 115 
,  relation  to  work,  399 

to  the  formation  of  glycogen, 
248 
,  calorific  value  of,  626,  628 
,  nutritive   value  of,  624—628,  645— 

650 
,  rancidity  of,  110 
,  absorption  of,  352 — 356 
,  behavior  with  gastric  juice,  304 
,  behavior  with  pancreatic  juice,  325 
,  saponification  of,  110,  332,  353 
,  action  of,  on  the  secretion  of  bile,  260 
,  action  of,  on  the  secretion  of  gastric 

juice,  304 
,  action  of.   on  the  secretion  of  pan- 
creatic juice,  322 
,  iodized,   behavior  of,  in  the  animal 

body,  372,  458 
,  estimation  of,  114,  447 
,  metabolism  of,  in  activity  and  at  rest, 

399 
,  metabolism  of,  in  starvation,  630 
,  metabolism  of,  with    various    foods, 
640,  645,  646,  652 
Fat  formation,  from  proteids,  372 — 374 


INDEX. 


683 


Pal  formation  from  carbohydrates,  31  1 
Fat-sweat,  694 

Fatty  acids,  general  properties,  detection 
and  occurrence,  108 — 115, 
:;hi 
,  solubility  in  bile,  332,  353 
,  absorption  of,  352 

,  synthesis,  375 

to  neutral  fats,  352, 
374 
Fatty  degeneration,  372 
Fatty  infiltration,  241 
Fatty  series,  behavior  of  members  in  the 

animal  body,  539 
Fatty  tissue,  370.  371 

.  behavior  with  gastric  juice, 
304 
Feathers,  5S 

,  pigments  of,  502 
Fehling's  solution,  94,  566 
Fellie  acid,  268 

Fermentation,  4,  10,  11,  88,  90,  93 
in  the  intestine,  333 
in  the  urine,  5>1,  5S4 
in  the  gastric  contents,  312. 
See  also  various  fermen- 
tations, alcoholic,  etc. 
Fermentation  lactic  acid,  properties,  oc- 
currence, etc., 
388,  389 
in     the     gastric 
contents,  298 
in  the  souring  of 
milk,  438 
,  detection  in  the 
gastric      con- 
tents, 315 
Fermentation  test  in  the  urine,  563,  569 
Ferments  in  general,  10 
inorganic,  15 
.     See  various  enzymes. 
Ferratin,  241 
Ferrine,  241 

Fevers,  elimination  of  ammonia  in,  534 
,  elimination  of  uric  acid  in,  186 
,  elimination  of  urea  in,  46S 
,  elimination  of  potassium  salts  in, 

533 
,  metabolism  of  proteids  in,  468 
Fibres,  elastic,  in  sputum,  615 

,  reticulate,  358 
Fibrin,  23,  26,  143,  145,  146,  153,  189,  192, 
193 
,  occurrence  in  transudates,  220 
,  Henle's,  419 
Fibrin  coagulation,  146—148,  1S9— 198 
Fibrin  calculi.  3  is.  5  6 
Fibrin  digesl  ion,  300,  313,  327,  329 
Fibrin  ferment,  13,  144,  146,  1S9— 198 
Fibrin  formation,  146-   lis,  iso—igs 
Fibrin  globulin,  14S.  153 
Fibrin  soluble.     See  Serglobulin. 
Fibrinogen,   24,  26,   144—148,   153,   192, 

193,224 
Fibrinolysis,  146 


Fibrinoplastic  substance.   See  Serglobulin. 
Fibroin,  26,  65,  (it; 
Fischer-Weidel's  reaction,  133 

Fish-bones,  368 

Fish-eggs,  27,  433 

Fish-scales,  (if),  133 

Fish,  bile  of,  •_'(•,•_> 

,  spermatozoa  of,  47,  128 

,  swimming-bladder  of,  133,  613 

,  visual   purple  of,    1 15 
Flesh,  metabolism,  in  starvation,  629 

,  metabolism,    with    various    foods, 
639—650 
Flesh  quotient,  404 
Florence's  sperms  reaction,  420 
Fluorine  in  bones,  366 

in  enamel,  370 
Fly-maggots,  formation  of  fat  in,  373 
Foods,  influence  on  the  secretion  of  intes- 
tinal juice,  317 
,  influence  on  the  secretion  of  bile, 

260 
,  influence  on  the  secretion  of  gastric 

juice,  296,  297 
,  influence  on  the  secretion  of  pan- 
creatic juice,  321 — 322 
,  influence  on  the  secretion  of  milk, 

456 
,  influence  on  the  elimination  of  uric 

acid,  4S5 
,  influence  on  the  elimination  of  urea, 

467,  46S 
,  influence    on    the    elimination    of 

xanthine  bodies,  494 
,  influence  on  faeces,  341,  348,  619 
,  influence  on  metabolism,  633 — 650 
,  various,  rich  in  protcid.  639 — 650 
,  various,  mixed,  639 — 650 
,  insufficient  supply  of,  633 — 639 
Foodstuffs,  necessity  of,  616 

,  combustion  heat  of,  624—628 
Formaldehyde,  formation  in  plants,  1 .  92 
,  action  upon  proteids.  .".7 
,  combination  with  urea.  171 
,  relation    to    sugar   forma- 
tion, 91 
Formic  acid   in  gastric  contents,  316 

,  passage  of,  into  urine,  520, 
539 
Frog's  eggs,  membrane  of,  51 
Fructose.     See  La^vulose. 
Fruit-sugar.     See  Lsvulose. 
Fundus  glands,  295.  307 
Fun^i.  glycogen  therein,  245 
Fumaric  acid.  23 
Furfuraervluric  acid,  545 
Furfurol  from  pentoses.  90 

,  relation  to  protcid  reactions,  31 
,  relation    to     IVttenkofer's    bile- 
acid  tests,  263 

,  reagenl  for  urea.  473 
,  behavior  in  the  animal  body,  545 
Fuscin,  416 

Galactonic  acid,  98 


684 


INDEX. 


Galactose,  85,  97,  101,  444 

,  from  cerebrins,  410,  411 
,  relation  to  formation  of  glyco- 
gen, 250 
Galactosamine,  56,  98 
Galactosides,  87 
Gallacetophenon,  behavior  in  the  animal 

body,  545 
Gallic  acid,  behavior  in  the  animal  body, 

511,  545 
Gallois's  inosite  test,  3S7 
Galtose,  87 

Gas,  exchange  of,  in  various  ages,  652,  653 
,  exchange  of,  through  the  skin,  596 
,  exchange  of,  in  starvation,  629,  630 
,  exchange  of,  in  various  conditions  of 

the  body, 399, 630, 633,  652,  656 
,  exchange  of,  in  the  muscles,  395,  399 
,  exchange  of,  abstinence  value  of,  632 
Gases  of  the  blood,  598 — 603 
of  the  intestine,  336 
of  the  bile,  277,  603 
of  the  urine,  536,  604 
of  the  hen's  egg,  433,  434 
of  the  lymph,  603 
of  the  milk,  448,  453 
of  the  muscles,  392,  395 
of  the  transudates,  219,  604 
Gastric  contents.     See  Chyme. 
Gastric  fistula,  295 
Gastric  juice,  295 

,  composition  of,  298 

,  secretion  of,  296,  297,  308 

,  estimation  of  acidity  of,  315, 

316 
,  relation  to  intestinal  putre- 
faction, 311,  340 
,  artificial,  264 

,  action  of,  300—308,  308— 
313,  442,  443,  451 
Gastric  lipase,  306 
Gastric  mucosa,  295 
Gelatine,  19,  20,  62,  80 

,  relation  to  glycogen  formation, 

247 
,  putrefaction  of,  334 
,  nutritive  value  of,  643,  644 
,  behavior  with  gastric  juice,  304 
,  behavior  with  pancreatic  juice, 
330 
Gelatine  and  the  detection  of  trypsin,  290 
Gelatine-forming  substances.     See  Colla- 
gen. 
Gelatine  peptones,  45,  63 
Gelatine  sugar.     SeeGlycocoll. 
Gelatinous  tissues,  359 
Gelatoses,  63 

,  relation  to  blood  coagulation, 
143 
Generation,  organs  of,  419 — 436 
Gentisic  acid,  512 

,  behavior  in  the  animal  body, 
545 
Gentisic  aldehyde,  512 
Gerhardt's  diacetic  acid  reaction,  576 


Globan,  34 
Globin,  49,  165 
Globulins,  26 

,  general  characteristics,  33 
,  in  urine,  550 
,  in  protoplasm,  117 
.    See  also  the  different  globulins. 
Globuloses,  39 
Glucalanin,  65 
Glucase,  155,  289,  292 
Glucocyanhydrin,  85 
Glucoheptose,  85 
Gluconic  acid,  84,  92 
Glucosamine,  51,  86,  98,  99,  589 
from  chitin,  5S9 
from  proteins,    23,   50,   53, 
424,  429,  432 
Gluconucleoproteids,  57 
Glucoproteids,  26,  51—55,  118,  430 

,  relation    to    formation    of 
glycogen,  250 
Glucoproteose,  43 
Glucosaminic  acid,  87 
Glucosan,  92 
Glucose.     See  Dextrose. 
Glucosides,  14,  87,  91 
Glucosoxime,  85 
Glucoron,  100 
Glucothionic  acid,  232,  361 
Glucuronic  acid,  99,  100 

,  relation  to  formation  of 

glycogen,  248 
,  conjugated,  99 
in  blood,  154 
in  bile,  262,  274 
in   urine,  521,  522,   541, 
546  572 
Glutamic  acid,  21,  24,  60,  61,  71 
Gluteines,  63 
Gluten  casein,  81 
Gluten  proteins,  81 
Glutin.     See  Gelatine. 
Glutin  peptones,  45,  63 
Glutokyrin,  45 
Glutolin,  132 
Glutose,  87 

Glycerine  aldehyde,  80 
Glycerine,  relation  to  formation  of  glyco- 
gen, 247 
Glycerophosphoric  acid,  120,  209,  232,  237, 

274 
Glycerophosphoric  acid  in  urine,  521,  525 
Glyceroses,  92 
Glycin.     See  Glycocoll. 
Glycocholeic  acid,  264 
Glycocholic  acid,  262,  264 

,  occurrence      in      excre- 
ments, 337 
,  occurrence  in  bile  from 

various  animals,  276 
,  absorption  of,  356 
,  behavior     to     intestinal 
putrefaction,  337 
Glycocholates  from  rodents,  265 
Glycocoll,  66 


INDEX. 


685 


Glvcocoll,  relation    to    formation    of   uric 
acid,  184,  188 
,  relation    to   fotmatiOD   of   urea, 

169,  540 
,  synthesis  with  glycocoll,  2,  500, 
"  543 
Glycogen,  15,  244—254 

,  origin  of,  247 — 255 

,  illation    to    muscular  activity, 

387,  395 
,  relation  to  muscle  rigor,  294 
,  occurrence  in  sputum,  015 
,  occurrence  in  leucocytes,  186 
,  occurrence  in  the  lungs,  61  I 
,  occurrence  in  the  lymph,  212 
,  occurrence  in   protoplasm,   117, 
123,  ISC,  227 
Glycolysis,  154,  259,  331 
I  rlycolytic  enzyme,  154,  331 
Glycosuria,  254—259 

,  alimentary,  255,  350 
'  rlycosuric  acid,  512 
Glycylalanin,  25,  45 

<  rlycylalanin  anhydride,  25 

<  rlycylglycin,  25 

Glyoxyl  diureid.     See  Allantoin. 
Glyoxylic  acid,  as  reagent,  31 
Gmelin's  test  for  bile-pigments,  271 

test   for  bile-pigments  in  urine, 
559 
Goat's  milk,  449,  450 
Gold  equivalent  of  the  proteids,  28 
Goose-fat,  absorption  of,  354 
Gorgon  in,  65 

Gout,  elimination  of  uric  acid  in,  485,  486 
Graafian  follicles,  422 
(irape-moles,  435 
Grape-sugar.     See  Dextrose. 
Guaiacum  blood  test,  555 
Guanine,  131,  133 

in  urine,  494 
Guanine  gout,  134 
Guano,  133,  131,  4S5 
Guano-bile  acids,  265 
Guanovulit.  133 
Guanylic  acid,  127,  128 
Gulonic  acid  lacton,  99 
Gulose,  91,  96 
Gums,  various,  105,  106 

,  animal,  52 

,  animal,  in  urine,  521 
Gunning-Lieben's  acetone  reaction,  575 
Giinzberg's  reagent  for  free  HC1,  314 

Hemagglutination,  163 

Hemataerometer,  008 

Hsematin,  175.  176 

,  relation  to  bilirubin.  270 
■  relation  to  urobilin,  516 

Hsematinogen,  1  s  1 

Hsmatinometer,  182 

Hematinic  acids,  177 

Haematinic  acid  amide,  177,  270 

Haernatochlorin,  435 

Haematocrit,  199 


Hematogcn,  427, 

Hematoglobulin.     See  ( ^xyhsemoglobin. 

lla-matoidin,  181 

,  relation    to    bilirubin,     181, 

269,  270,  280 
,  occurrence  in  sputum,  615 
,  occurrence  in  corpora  Lutea, 

422 
,  occurrence    in    excrem 

342 
,  occurrence  in  sediment 
Hsematoporphyrin,  179 

,  relation    to    bilirubin, 

180,  270 
,  relation    to    urobilin, 

180,  516 
,  occurrence    in    urine, 

514,  556 
,  occurrence     in    lower 
animals,  592 
ILematoscope,  183 
{hematuria,  554 
Haemerythrin,  184 
Ihemin,  178 

Ibemin  crystals,  178,  556 
Haemochromogen,  165,  175,  176 

,  occurrence  in  muscles, 
382 
Haemocyanin,  184 
Haemoglobin,  26,  50,  170 

,  composition  of,  166 

,  properties  and  behavior,  170 

,  quantity     in     blood,     165, 

203—208 
,  quantitative         estimation, 

182,  183 
,   See    also    oxyhemoglobin 
and  the  combination-  of 
haemoglobin    with    other 
gases. 
Hemoglobinuria,  555 
Haemolysis,  162 
Haemolysins,  156.  162 
Ibemometer,  183 
Haemopyrrol,  165 
Btemorrhodin,  174 
Haemoverdin,  174 
Basel's  coefficient,  537 
Hair,  5S.  5ss 

.  pigments  of,  592 
Hair-balls.  344 
Half-rotation.  88 
Hammarsten's  reaction  for  bile-pigments, 

271.  559. 
Haptogen-memhrane.  439 
Heat,  action  of.  on  metabolism.  652,  657 
of  combustion  of  various  foodstuffs, 
624— 628 
,  loss  of.  through  the  skin,  597,  657 
generated  in  plants.  2 
Helicoproteid.  55 
Heller's  albumin  test.  30 

albumin  test  applied  to  urine,  548 
Heller-Teichmann's  blood  test,  556 
Hemicelluloses,  107 


686 


INDEX. 


Hemicollin,  63 
Hemielastin,  60 
Hemipeptone,  39 
Hemp-seed  calculi,  5S5 
Hen's  egg,  426—436 

,  incubation  of,  433,  434 
Heptoses,  84 

Herring,  spermatozoa  of,  47,  48 
Heteroproteoses,  39,  40,  43,  46,  552 
Heterosyntonose,  80 
Heteroxanthine,  131 

in  urine,  495 
Hexaglycylglycinethyl  ester,  330 
Hexobioses,  101 
Hexon  bases,  21,  77—80 
Hexoses,  91—98 

from  nucleoproteids,  51 
from  nucleic  acids,  127 
.  See  also  the  various  hexoses. 
High  altitude,  action  on  the  blood,  207 
Hippokoprosterin,  284 
Hippomelanin,  591 
Hippuric  acid,  500 

,  properties    and    reactions, 
502 
estimation  of,  502 
,  formation  in  the  body,  2, 

500,  501,  543 
,  cleavage  of,  500,  502 
,  occurrence  of,  500 
as  sediment,  5^3 
Histidin,  21,  24,  43,  48,  79 
Histons,  19,  28,  47,  48,  80,  196,  229 

in  urine,  554 
Histozyme,  503 
Hofmann's  tyrosin  test,  73 
Holothuria,  mucin  of,  54 
Holozvm,  195 

Homocerebrin,  408,  409,  410 
Homogentistic  acid,  8,  73,  506,  511—513 
Hoppe-Seyler's  CO  blood  test,  173 
xanthine  test,  133 
Horn,  57,  588 
Horn  substance  in  the  gizzard  of  birds,  59 

See  also  Keratins. 
Huckleberries,  coloring  matter  of,  in  urine, 

.-,17 
Human  milk,  450 — 455 

,  behavior    in    the    stomach, 
451 
Humirj  substances  in  urine,  514 
Humor,  aqueous,  224 
,  vitreous,  416 
Huppert's  reaction  for  bile-pifjments,  271 
reaction    for    bile-pigments    in 
urine, 559 
Hyalines,  53 

of  the  walls  of  hydatid  cysts,  590 
of  Hovida's  substance,  118,  186, 
227 
Hyalogens,  53 
Hyalomucoid,  416 
Hydatid  cyste,  590 
Hydraemia,  208 
Hydramnion,435 


Hydrazins,  85 
Hydrazons,  85 
Hydrobilirubin,  270 

,  relation  to  urobilin,  516 
Hydrocele  fluids,  220,  223 
Hydrocephalus  fluid,  224 
Hydroquinone,   8,   506,   547 
Hydroquinone  sulphuric  acid,  503,  506 
Hydrochloric  acid,  secretion    in   stomach, 
298,  306,  313 
,  anti-fermentive  action 

of,  312 
,  action  of,  on  secretion 
of  pancreatic    juice, 
322 
,  action  of,  on  pylorus, 

309 
.quantitative  estimation 
in    gastric  contents, 
315,  316 
,  reagents  for  free  HC1 
in    gastric   contents, 
314 
Hydrocinnamic  acid,  behavior  in  the  ani- 
mal body,  500 
Hydrocyanic  acid,  action  on  peptic  diges- 
tion, 303 
,  action    on    tryptic    di- 
gestion, 328 
Hydrogen  in  putrefactive  and  fermentive 

processes,  4,  334,  336 
Hydrogen    peroxide,     decomposition    of, 

by  catalases,  6,  13 
Hydrogenases,  13 
Hydrolytic  cleavages,  10 

.   See  also  the  various 
cleavages. 
Hydronephrosis  fluid,  462 
Hydroparacoumaric  acid,  511 

,  in       intestinal 
putrefaction, 
334 
Hydroxylamine,  poisoning  with,  499 
Hyoglycocholic  acid,  265 
Hyperglycemia,  255,  256 
Hyperistonic  solutions,   162 
Hypisotonic  solutions,  162 
Hypnotics,  relation  to  formation  of  gly- 
cogen, 248 
Hypogaic  acid,  115 
Hyposulphites  in  the  urine,  524 
Hypoxanthine,  131 

,  properties,  134,  135 
,  passage    of,    into    urine, 
494 

Tchthidin,  427,  433 
Ichthin.  433 

Ichthulin,  26,  55,  427,433 
Ichthvlepidin,  65 
Icterus,  280,  281 

in  urine,  558 
Immunity,  17,  156 
Incubation  of  the  egg,  433 — 435 
Indican  test,  Jaffa's,  509 


INDEX. 


687 


Indican  test,  Obermeyer's,  50$ 

Indican,  urine.  507      ">(l^ 

,  elimination    in    starvation,    337, 

507 
,  elimination  in  disease,  507 
Indigo,  507 

in  sweat,  596 
in  urinary  sediments,  5S3 
[ndigo  blue,  335,507,  514 
Indol,  properties,  335 

,  formation  from  proteid,  21,  22,  23 
,  formation  in  putrefaction,  334,  335, 

507,  510 
,  formation  from  melanins,  592 
Indophenol  blue,  8 
Indoxyl,  335,  507 

Indoxyl-glucuronic  acid,  507,  509,  522 
Indoxyl  red,  508 
Indoxyl-sulphurie  acid,  507,  508 
Inosimc  acid.  128,  383,  3S6 
Inosite,  properties   and   occurrence,    386, 
387 
,  in  urine,  573 

,  relation  to  formation  of  glycogen, 
245 
Integral  factor,  490 
Intestinal  calculi,  343 
Intestinal  contents,  331 — 340 
Intestinal  fistula,  317 
Intestinal  gases,  334,  336 
Intestinal  juice,  316—319 

enzymes  of,  318,  321,322 
Intestinal  mucosa,  316 
Intestine,  putrefactive  processes  in,  333 — 
340,  501,  503,  507 
,  reaction  in,  333,  340 
,  absorption  in,  339,  344 — 357 
,  digestive  processes  in,  331 — 334 
Intracellular  enzyme  action,  11 
Inulin,  104 

,  relation  to  formation  of  glycogen, 
247 
Inversion,  101,  318,  349 
Invertases,  13.  14,  102;  318,  349 
Invert-sugar,  101 

Iodides  and  Becretion  of  gastric  juice,  307 
Iodine  equivalent,  1 14 
Iodine,  passage  of,  into  milk,  459 
,  passage  of,  into  sweat,  596 
,  passage  of,  into  saliva,  293 
Iodized  proteids,  22,  65,  66,  234 
Iodized  fats,  372.  L58 
Iodo-cholalic  acid  compound,  266 
Iodoform,  behavior  in  the  animal  body, 
540 
test,  Gunning's,  575 
test,  Lieben's,  575 
Iodogorgonic  acid,  65 
Iodonsernatin,  179 
Iodospongin,  65 
Iodothvreoglobulin,  234 
Iodothvrin,  231,  234,  236 
Ion  action.  28,  140.  141,  164 

in  blood-serum,  157 
in  glands,  231,  234 


Iron  in  blood,  156,  201 

in    blood-pigments,    166,   176,    179, 
182,  280 

in  bile,  262,  274,  280 

in  hair.  .^>.  592 

in  urine,  535 

in  the  liver,  210,  242,  243,  281 

in  milk,  -lis,  455,  459 

in  the  spleen,  232,  233 

in  muscles,  391,  402 

in  new-born,  233,  243,  455 

in  protein  substances,  8,  18,  34,  125, 
232,  241,  281 

in  cells,  140 

,  elimination  of,  274,  281,  294,  535 

and  blood  formation,  127 

and  bile  formation,  280 

,  absorption  of,  206,  207 
Iron  salts,  elimination  by  the  urine,  535 
,  action  on  the  blood,  207 
,  absorption  of,  207 
Iron  starvation,  637 
Ischuria  in  cholera,  597 
Isobilianic  acid,  266 
Isocholanic  acid,  267 
Isocasein,  441 
Isocholesterin,  284,  285 

in  vernix  caseosa,  593 
Isocreatinine,  3S4 
Isodynamic  law,  626 
Isoglucosamine,  86 
Isomaltose,  102,  291,  325 
in  urine,  521 
Isosaccharin,    relation    to    formation    of 

glycogen,  248 
Isotonic  solutions,  162 
Isotropous  substance,  376 
Ivory,  370 

Jaffa's  indican  test,  508 

creatinine,  4^3 
Janthinin,  593 
Japanese,  nutrition  of,  660 
Jaune  indien,  100 
Jecorin,  123,  232,  242 

,  in  blood,  154 
Jequirity  bean,  17 
Jolles's  reaction  for  bile-pigments,  560 

Kephir,  445,  449 

,  anti-putrefactive  action,  338 

Kerasin,  408,  409,  410 

Keratose,  58 

Keratins,  26,  57,  58,  59 

,  behavior  in  the  stomach,  304 
,  behavior  with  pancreatic  juice, 
330 

Ketones,  behavior  in  the  animal  bodv,  541 

Ketoses,  ^4,  97 

Kidneys.  461,  462 

,  relation  to  formation  of  urea,  472 
,  relation  to  formation  of  hippuric 
acid,  501 

Kinases,  321 


6S8 


INDEX. 


KjeldahFs  method  of  determining  nitro- 
gen, 475 

Knapp's  titration  method,  56S 

Knee-joint  cartilage,  364 

Knop-Hiifner's  method  for  determining 
urea,  4S0 

Koprosterin,  284,  341 

Kumyss,  445,  449 

Kyestein,  5S3 

Kynurenic  acid,  511,  513,  526 

Kyrin,  45 

Laborer,  diet  of,  659-665 

Lactalbumin,  26,  343,  344 
Lactase,  318,  349 

Lactates.     See  Lactic  acids,  also  390 
Lactic-acid  fermentation,  88,  94,  312,  313, 
331,333,388, 
438,  445 
in  intestine,  331, 

333 
in  stomach,  312, 

313 
in  milk,  438,  445 
Lactic  acids,  438 — 441 

in  intestine,  331,  333 

in  urine,  3S8,  488,  521 

in  bones,  369 

in  stomach,    298,  314,   315, 

316 
,  relation  to  formation  of  uric 

acid,  488 
.    See  also  Paralactic  and  Fer- 
mentation lactic  acids. 
Lacto-caramel,  445 
Lacto-globulin,  443 
Lactones  of  varieties  of  sugars,  84 
Lactophosphocarnic  acid,  444 
Lactoprotein,  444 
Lactose.     See  Milk-sugar,  103,  444 
Larvolactic  acid,  388 
Lsevulose,  84,  86,  91,  92,  96,  97 
in  urine,  570 
in  blood,  154 

relation  to  glycogen  formation, 
250 
,  absorption  of,  349,  350 
,  behavior  in  diabetics,  256 
in  transudations,  220 
Lsevulosemethyl  phenylosazone,  97 
.  Laiose,  570 
Lake  color  of  blood,  189 
Lanoceric  acid,  594 
Lanolin,  285 
Lanopalmitic  acid,  594 
Lanugo  hair,  435 
Lard,  absorption  of,  354 
Large  intestine,  extirpation  of,  357 

,  secretion  of,  319 
Latebra,  426 
Laurie  acid,  108 

in  butter,  440 
in  spermaceti,  115 
Lead  colic,  elimination  of  urobilin  in,  517 


Lead  in  the  blood,  201 
in  the  liver,  244 
,  passage  of,  into  milk,  459 
Lecithalbumins,  35,  295 

,  relation    to    secretion   of 

gastric  juice,  295 
,  relation   to    secretion   of 
urine,  463 
Lecithin,  120 

in  milk,  440,  452 
in  the  liver,  242 
,  importance  for  cells,  637 
,  putrefaction  of,  122,  337 
Legal's  acetone  reaction,  575 
Leinolic  acid,  108 
Lens  (see  Crystalline  lens),  416 
,  capsule  of,  53,  416,  417 
,  fibres  of,  417 
Leo's  sugar,  570 
Lepidoporphyrin,  593 
Lepidotic  acid,  593 
Lethal,  115 
Leucaemia,  blood,  131,  208,  209 

,  uric  acid,  elimination  in,  234, 

485,  486 
,  xanthine  bodies  in,   131,  208, 
494 
Leucin,  68 — 70 

,  relation  to  formation  of  uric  acid, 

488 
,  relation  to  formation  of  sugar,  251 
,  relation  to  formation  of  urea,  469, 

539 
,  preparation,  69,  70 
,  passage  of,  into  urine,  579 
,  behavior  in  the  animal  body,  251, 
469,  539 
Leucin  ethylester,  70 
Leucinic  acid,  69 
Leucinimid,  70 

Leucocytes,  relation  to  absorption,  347 
,  relation  to  formation  of  uric 
acid,  487 
in  thymus  gland,  229,  231 
Leucomaines,  17 

in  urine,  526 
in  muscles,  386 
Leuconuclein,  192,  229 
Levulinic  acid,  52,  90,  444 
Lichenin,  105 

Lieben's  acetone  reaction,  575 
Lieberkiihn's  alkali-albuminate,  35 

glands,  317 
Liebermann's  reaction  for  proteids,  31 
Liebermann-Burchard's  reaction  for  cho- 

lesterin,  284 
Liebig's  titration  method  for  estimating 

urea,  476—478 
Lienoproteases,  232 
Ligamentum  nucha?,  59,  359 
Lignin,  107 
Linoleic  acid,  108,  112 
Linseed-oil,  feeding  with,  371,  458 
Lion's  urine,  485 
Lipanin,  absorption  of,  354 


IXhl.X. 


Lipase  1  1 

in  blood,  155 
in  stomach,  306 
in  pancreatic  juice,  325 
in  milk.   I  1  1 
Lipiawsky's  aoetoacetio  acid  reaction,  577 
Lipochromea,  1  "><>,  428 
Lipoids,  L20 
Lipuria,  579 
Lithium  in  blood,  201 
Lithium  lactate,  390 
Lithium  urate,  491 
Lit  1 1 1 > I > i  1  i < -  acid,  344 
Lithofeuio  acid,  268,  344 
Lithuric  acid,  526 
Liver,  239—244 

,  relation  to  coagulation  of  blood,  197 
,  relation  to  formation  of  uric  acid, 

ls7,  4S8 
,  relation  to  formation  of  urea,  469, 

470,  471.  472 
,  blood  of,  203,  252,  253 
,  proteids  of,  240 
,  fat  of,  241 

,  quantity  of  sugar  in,  253 
Liver  atrophy,  acute  yellow,  277,  579 

,  elimination  of  ammonia  in, 

472 
,  elimination  of  urea  in,  472 
,  elimination  of  leucin  and 

ty rosin  in,  579 
,  elimination  of  lactic  acid 
in,  388,  521 
Liver  cirrhosis,  ascitic  fluid  in,  224 

,  action  of,  on  the  elimina- 
tion  of    ammonia    and 
urea,  472 
Liver  extirpation,  elimination  of  ammonia 
with,  472,  4S7 
,  elimination  of  uric  acid 

with,  487 
,  elimination     of     lactic 
acids  with,  388,  487, 
521 
,  action  on  formation  of 
bile,  278 
Lotahiston,  49 
Lung  catheter,  608 
Lungs,  614,  615 
Luteins,  156,  428 

in  corpora  lutea,  181,  422 
,  egg-yolk.  428 
in  serum,  156 
,  relation  to  haematoidin,  181,  428 
Lymph,  211—217 
Lymphagogues,  215 
Lymphatic  glands,  229 
Lymph-cells,  quantitative  composition  of, 
231 
.     See  also  Leucocytes. 
Lymph-fibrinogen.    See  Tissue-fib rinogen. 
Lysabinic  acid,  37 
Lysatin  and  lysatinin,  79 
Lysin,  48,  78,  79 
Lysins,  l7 


Lysuric  acid,  79 

Mackerel,  flesh  of,  403 

,  sperm  of,  47,  l'» 

Madder,  feeding  with,  363 

Magnesium  in  urine,  529,  535 
in  bones,  366,  370 
in  muscles,  391,  W2,  404 

.     See  also  various  tissues  and 
fluids. 
Magnesium  phosphate  in  intestinal  calculi, 
li:; 
in  urine,  529,  535 
in    urinary    calculi, 

584,   585 

in      urinarv      sedi- 
ments, 5S2,  583 
in  bones,  366,  370 
Magnesium  soaps  in  excrements,  341 
Malic  acid,  behavior  in  the  animal  body, 

539 
Maltase,  14,  318 
Maltodextrin,  105 
Maltoglucase,  14,  155 
Maltose,  102 

,  formation  from  starch,  102,  104 

291,  325 
,  absorption  of,  349,  350 
,  relation  to  glycogen  formation, 
250 
in  intestine,  332,  349,  350 
,  occurrence  in  urine,  572 
Mammary  glands,  437,  457 
Mandelic  acid,  543 
Man  in  poorhouse,  diet  of,  664 
Mannite,  85 

,  relation  to  format  ion  of  glycogen, 
247 
in  urine,  526 
Mannonic  acid,  92 
Mannose,  88,  91,  92,  96 
Mare's  milk,  449,  450 
Margarine  and  margaric  acid,  111 
Marsh-gas,  formation  in  putrefaction,  21, 

334,  336 
Maschke's  creatinine  reaction.  482 
Meat  extracts,  action  on  secretion  of  gas- 
tric juice,  298 
Meat,  utilization  in  intestinal  tract,  348 
,  calorific  value  of,  627,  628 
,  digestibility  of,  310 
,  composition  of,  373,  401-404 
.     See  also  Muscles. 
Meconium,  342 
Medulla  oblongata,  412 
Melanins,  591,  592 

in  the  eye,  416 
in  the  urine,  557 
Afelanogen  in  the  urine,  557 
Melanoidie  acid,  591 
Melanoidins,  19,  21,  23,  592 
Melanotic  sarcoma,  pigment  of,  591 
Melissvl  alcohol,  115 
Membranines,  53,  364,  416 
Menstrual  blood,  204 


690 


INDEX. 


Menthol,  behavior  in  the  animal  body,  546 
Mercaptan,  from  proteins,  21,  24,  58 
Mercapturic  acid,  76,  546 
Mercury  salts,  passage  of,  into  milk,  459 
,  passage  of,  in  sweat,  596 
,  action  on  ptyalin,  292 
,  action  on  trypsin,  329 
Mesitylene,  behavior  in  the  animal  body, 

543 
Mesitylenic  acid,  543 
Mesoporphyrin,  180 

Metabolism,  dependence  of  external  tem- 
perature upon,  657 
in  various  ages,  654 
in  work  and  rest,  394 — 401, 

655—657 
in  different  sexes,  653 
in  starvation,  628—633 
with      different      foodstuffs, 

639—652 
in  sleep  and  waking,  657 
,  calculation  of  extent  of,  621 — 
625,  632 
Metalbumin,  423 
Metallic  soles,  15 
Metaphosphoric  acid,  as  reagent  for  pro- 

teids,  30,  549 
Metazym,  195 
Methtemoglobin,  171,  183 

in  urine,  555 
Methal,  115 
Methane,  formation  in  putrefaction,  21, 

334, 336 
Methose,  92 
Methylenitan,  91 
Methyl  glycocoll.     See  Sarcosin. 
Methylguanidine,  384,  482 
Methylguanidin-acetic    acid.      See    Crea- 
tine. 
Methylhydantoic  acid,  540 
Methyl  indol.     See  Skatol. 
Methyl  mercaptan  in  proteids,  21,  334,  336 
Methyl  pentose.     See  Rhamnose. 
Methvl  pvridine,  behavior  in  the  animal 

body,  543 
Methyl-pyridyl-ammonium        hydroxide, 

:.I7 
Methyluramine,  3S4,  482 
Methyl  xanthine,  131,  495 
Micrococcus  restituents,  346 
Micrococcus  ureac,  581 
Micro-organisms   in    intestinal   tract,    16, 

312,  333,  337,  341 
Milk,  437,  HO 

,  secretion  of,  457 

,  consumption   of,   in   intestine,   348, 

355,  451 
,  blue  or  rod,  460 

,  anti-putrefactive  action  in  intestine, 
338,  504 
in  disease,  459 
,  passage  of  foreign  bodies  into,  459 
,  behavior  in  the  stomach,  309,  311, 

451 
.  See  also  different  varieties  of  milk. 


Milk-fat,  440,  450 

,  formation  of,  458 
Milk-globules  from  cow's  milk,  439,  440 

from  human  milk,  450 
Milk-plasma,  440 
Milk-sugar,  103,  444 

,  relation  to  formation  of  gly- 
cogen, 247 
,  properties,  444,  445 
,  fermentation,  438,  445,  449 
,  calorific  value  of,  625 
,  quantitative  estimation,    446, 

448 
,  absorption  of,  350 
,  passage  of,  into  urine,  250,  570 
,  origin  of,  459 
Millon's  reagent,  30 

Mineral  acids,  alkali-removing  action   of, 
464,  534,  602,  635 
,  action  on  the  elimination 
of  ammonia,  464,  534 
Mineral  bodies,  elimination  in  starvation, 
431,  533 
,  insufficient  supply  of,  634, 

636 
,  behavior  in  the  organism, 

139,  140,  634,  635 
.  See  also  the  various  fluids, 
tissues,  and  juices. 
Mixture  of  the  nitrogenous  substances  in 

the  urine,  468,  485,  486 
Modified  proteids,  29 
Molisch's  sugar  test,  95,  565 
Monamino  acids,  66 — 77 

,  behavior  in  animal  body, 
469,  539 
Monosaccharides,  84 — 99 
Moore's  sugar  test,  93 
Morner-Sjoqvist's  method    of    estimating 
urea,  479 
method    of    estimating 
acidity,  316 
Morner's     tyrosin     test,     73.     See     also 

Deniges. 
Morner's  reaction  for  acetoacetic  acid,  512 
Morphine,  passage  of,  into  urine,  522,  547 

,  passage  of,  into  milk,  459 
Mucic  acid,  98,  105,  444 

,  relation  to  formation  of  gly- 
cogen, 248 
Mucilages,  vegetable,  105 
Mucin,  26,  51—54 

in  sputum,  615 
in  cysts,  425 
in  urine,  525,  553 
in  salivary  glands,  286,  288 
Mucin-like  substances  in  bile,  262,  277 
in  urine,  525,  553 
in  kidneys,  462 
in     thyroid    gland, 

235 
in     synovial    fluid, 


225 


Mucin  ogen,  51,  287 
Mucinoids.     See  Mucoids, 


INDEX. 


091 


■Mucin  peptone,  52,  304 
Mucoids,  26,  51,  53 

in  ascitic  fluids,  222 

in  the  vitreous  humor,  416 

in  the  cornea,  364 

in  connective  tissue,  358,359,305 

in  the  hen's  egg,  431 

in  cysts,  123,  I-  1 
Mucous  glands,  52,  286,  295 
Mucous  membranes  of  the  stomach,  295 

Mucous  tissue,  359 

Mucus  of  the  bile,  262,  276 

of  the  urine,  462,  525,  554 
of  synovial  fluid,  225 
Mulberry  calculi,  oSo 
Murexid  test,  491 

Muscle,  coagulation  of,  377,  380,  381,  393, 
394,  404 
,  permeability  of,  392 
Muscle-fibres,  376 
Muscle-pigmente.  382 
Muscle-plasma,  :<77,  378 

.coagulation  of,  378,  380, 
3 si,  382,  393,  394,  404 
Muscle  rigor,  393,  394 
Muscle-serum,  377 
Muscle-si  roma,  380 
Muscle-sugar,  388 
Muscle-sy ntonin,  380 
Muscles,  non-striated,  404 
,  striated,  376—404 
,  Mood  of.  201,  395,  399 
,  chemical   processes  iu  work  and 

rest,  391-101,  656 
,  chemical  processes  in  rigor,  393 
,  proteids  of.  377—383,  404 
,  extractives  of,  383— 393 
,  enzymes  of,  382 
,  pigments  of,  382 
,  fat  of,  391,  399,  403 
,  gases  of,  392,  395 
,  calorific  value  of,  625,  626,  627 
,  mineral  bodies  of,  391,  404 
,  amount  of  water  in,  403 
,  composition  of,  401 
Muscular  energy,  origin  of,  400,  401 
Musculamine,  385 

Muscular  force,  chemical  processes  in  mus- 
cles, 394 — 101 
,  action  of,  on  urine,  463, 

481,484,520 
,  action  of,  on  metabolism, 
395—401,  655—657 
Musculin,  379,  381,  403 
Mussels,  glycogen  of,  244 

,  muscles  of,   11)  I 
Mutton-fat,  feeding  with,  371 

,  absorption  of,  354,  355 
Mveline  forms,  405 
Mvelines,  408 
Myoalbumin,  378,  380 
Myogen,  381,  382 
Myogen  fibrin,  381,  393,  394 
Myoglobulin,  378,  380 
Myohsematin,  382 


Myoprotcid,  382 

Myosin,  24,  26,  373,  378,  379 

,  absorption  of,  '■'•  15 
Myosin  ferinei, t,  :;sl,  :;sj 
Myosin  fibrin,  379,  381 
Myosinogen,  381 
Myosinoses,  39 
Myricin,  1 15 

Myricvl  alcohol,  115 

Myristic  acid  in  animal  fat,  106 

in  butter,    1  ID 
in  bile,  274 
in  wool-fat,  59  I 
Myxedema,  235 

Myxoid  cysts,  122 

Nails,  57,  588 

Naphthalene,  action  on  urine,  547 

,  behavior  in  the  animal  body, 
542,  546 
Naphthol-glycuronic  acid,  547 
Naphthol,  reagent  for  sugar,  95,  565 

,  behavior  in  the  animal  body, 
522,  547 
Narcotics,  relation  to  glycogen  formation, 

248 
Native  proteids,  29 
Navel  cord,  mucin  of,  51,  52,  361 
Negative  phase,  196 
Neossin,  53 
Nerves,  107,  408,  413 
Xeuridine,  408,  412,  426 
Neurine,  120 

,  in  suprarenal  capsule,  237 
Neurochitin,  413 
Neuroglia,   107 
Neurokeratin,  ">7,  407,413 
Neutral  fats.     See  Fats. 
Nitrates  in  the  urine,  533 
Nitric-oxide  haemoglobin,  174 
Nitrites,  behavior  in  the  animal  body,  540 
Nitro-benzaldehyde,  behavior  in  the  ani- 
mal body.  5  1  I 
Nitro-benzoic  acid,  544 
Nitro-benzyl  alcohol,  546 
Nitro-eellulose,  107 
Nitro-hippuric  acid,  5  I  1 
Nitro-phenyl-propiolic    acid,  reagent    for 

BUgar,  82, 
565 
,  behavior  in 
the  animal 
bodv,  507, 
.-ii'.)' 
Nitro-toluene,    behavior    in    the    animal 

body, 546 
Nitrogen,  combined,  amount  of,  in  intesti- 
nal      evacuations, 
618 
,  in  meat.    103,  620 
,  in  urine,  468 
,  estimation        of,    in 
urine,  475 — 180 
Nitrogen  elimination  in  work    and    rest, 
396,  39S,  Qq5j  OjO. 


692 


INDEX. 


Nitrogen  elimination  in  starvation,  629, 
630 
with  various   foods, 

639—650 
by  the  intestine,  348, 

618 
by   the   urine,    468, 
530,  532, 618—620 
by     the     epidermis, 

619 
by  the  sweat,   596, 

619 
,  relation  to  the  elim- 
ination of    phos- 
phoric acid,  530, 
620 
,  relation  to  the  elim- 
ination    of     sul- 
phuric acid,  532, 
620 
,  relation  to  digestive 
activity,  534,  619, 
642 
Nitrogen,  free,  in  blood,  598 

,  in  intestine,  336 
,  in  stomach,  308 
,  in  secretions,  603 
,  in  transudations,  604 
,  in  urine,  536 
Nitrogen  in  the  proteid  molecule,  18,  19 
Nitrogenous  deficit,  619 
Nitrogenous  equilibrium,  619 

,  w  i  t  h      various 
foods,  640, 
641,  645,  648 
Nitroso-indol  nitrate,  335 
Non-striated  muscles,  404 
Norisosaccharic  acid,  98,  427 
Nubecula,  462,  581 
Nucleic  acids,  56,  57,  124,  125,  126—130 

in  the  urine,  554 
Nuclein  bases,  130 — 137 

in  blood,  131,  156 
in  the  urine,  494 
Nucleins,  56,  125,  126 

,  relation    to    elimination    of    al- 

loxuric  bases,  494 
,  relation    to    formation    of    uric 

acid,  486,  487 
,  relation  to  elimination  of   P205, 

529,  530 
,  behavior  with  gastric  juice,  56, 

125,  304 
,  behavior  with  pancreatic  juice, 
329 
Nuclein  plates,  186 
Nucleoalbumins,  26,34, 117, 126 

in  the  bile,  262,  278 
in  the  urine,  553 
in  the  kidneys,  462 
in  protoplasm,  117 
in  transudates,  218,  221 
,  behavior  in  pepsin  diges- 
tion, 29,  34,  35,  126, 
443,  451 


Nucleoglucoproteids,  56 
Nucleohiston,  48,  186,  229,  230 

,  relation  to  coagulation  of 
blood,  192 
in  urine,  554 
Nucleoproteids,  26,  50,  56,  57,  124,  125 
in  the  liver,  240 
in  gastric  juice,  298,  300 
in  blood,  148 
in  bile,  278 

in  mammary  glands,  437 
in  muscles,  380,  404 
in  the  kidneys,  462 
in  the  pancreas,  124,  125, 

320 
in  protoplasm,  118 
in  cell  nucleus,  118,  124 
■  in  thyroid  gland.  234 

in  thymus,  229 
,  behavior  in  pepsin  diges- 
tion, 56,  125,  304 
,  behavior  with  pancreatic 
juice,  329 
Nucleon,  385 

in  milk,  444 
Nucleosin,  138 
Nutrition  requirements,  640 

of  man,  659 — 666 
Nylander's  reagent.     See  Almen-Bottger's 
sugar  test. 

Obermeyer's  indican  test,  508 

Obermuller's  cholesterin  reaction,  284 

Odoriferous  bodies  in  the  urine,  547 

(Edema,  subcutaneous,  fluid  from,  225 

Oertel's  diet  cure,  665,  666 

(Esophageal  fistula,  296 

Oleic  acid,  112,  241 

Olein,  111 

Oligsemia,  207 

Oligocythemia,  207 

Oliguria,  537 

Olive  oil,  absorption  of,  354 

,  action  on  the  secretion  of  bile, 
261 
Onuphin,  53 
Oocyanin,  432 
Oorodein,  432 
Opalisin,  444,  452 
Opium,  459 
Optograms,  415 
Orcin  test,  90,  572 
Organic  acids,  behavior  in  the  animal  body, 

464,  534,  539 
Organized  proteids,  641,  642 
Organs,  loss  of  weight  in  starvation,  632 
Organs  of  generation,  419 — 436 
Ornithin,  24,  78,  543,  545 
Ornithuric  acid,  78,  543 
Orthonitrophenylpropiolic  acid.   See  Nitro 

phenylpropiolic  acid. 
Orylic  acid,  444 
Osaminic  acid,  86 
Osamines  of  varieties  of  sugar,  86 
Osazones,  86 


INDEX. 


693 


Osmosis,  relation  to  absorption,  357 

,  relation    to    lymph     formation, 
216 
Osmotic  pressure  of  blood,  158,  159 

of  urine,  465 
Osone,  86 
Ossein,  61,  365 

talbumoid,  365 

<  (rooomaooidi  52,  365 

I  iBteomalacia,  368,  369 
Osteoporosis.     See  Osteosclerosis. 

<  Isteosclerosis,  36S 
Otoliths,  lis 
Ovalbumin,  20,  23,  430 
(  )varian  cvsts,  422,  425 
Ovaries,  422 
Ovoglobulin,  23,  429 
Ovomucoid,  53,  431 
Ovovitellin,  24,  26,  326 
Ovum,  426 — 136 

< Oxalate  calculi,  585 

( Kalate  of  lime.     See  Calcium  oxalate. 

Oxalates,  action  on  blood  coagulation,  143, 

189 
Oxalic  acid,  origin,  20,  23,  498 

in  the  urine,  497,  582 
,  behavior  in  the  animal  body, 
498,  508,  539 
Oxalic  acid  diathesis,  498 
Oxaluria,  498 
Oxaluric  acid,  4S4,  497 
Oxamide,  21 

Oxidases,  7.  8,  13,  73,  155,  444 
Oxidation  ferment.     Sec  Oxidases. 
Oxidations,  1—9,  168,  169,  256,  334,  375, 

470.  507,  516,  539,  542,  604 
Oximes,  85 
Oxonic  acid,  485 

Oxyacids,  formation  in  putrefaction,  22, 
334 
,  detection  of,  511 
,  passage  of,  in  urine,  334,  511 
in  the  sweat,  596 
Oxvbenzoic  acid,  behavior  in  the  animal 

body,  443 
Oxybenzenes,  542 
Oxybutyrie  acid,  577 

,  detection    and    estima- 
tion, 578 
in  the  blood,  603 
,  passage    of,     into      the 
urine,  534,  573,  574 
Oxyethylsulphonic  acid,  behavior  in  the 

animal  body,  541 
Oxyfatty  acids  in  animal  fat,  108 
Oxygen,  consumption,  607 

in  work  and  rest, 

395,  399 
in  starvation,   630, 

633 
bv  the  skin,  596 
Oxygen,  activity  of,  3—9,  169 

in  the  blood.  599,  605 — 610 
in  the  intestine,  336 
in  the  lymph,  212,  603 


Oxygen   in  the  stomach,  308 

in     the      swimming-bladder     of 

fishes,  613 
in  secretions.  <>03,  60 1 
in  transudations,  604 
,  ten-ion  of,  in  blood,  604 — 610 
,  lack  of,  action  on  proteid  destruc- 
tion, 3S9,  397,  4 (is 
,  lack  of,  action  on  elimination  of 

lactic  acid,  389,  521 
,  lack  of,  action  on  elimination  of 

sugar,  389 
,  capacity,  628 
Oxygen-carriers,  7,  169 
Oxygen,  calorific  value  in  the  combustion 

of  different  foods,  623,  624 
Oxygen  consumption  in  the  blood,  171,  599 
Oxygenases,  8 
Oxyhaematin,  176 
Oxyhaemocyanin,  184 
Oxy  haemoglobin,  167 

,  dissociation  of,  167,  605 
,  properties  and  reactions, 

167—170 
,  quantitv  of,  in  the  blood, 

165,  200,  203—207 
,  quantitv  in  the  muscles, 

382 
,  passage  of,  into  the  urine, 

554 
.behavior     with    gastric 

juice,  304 
,  behavior   with    trvpsin, 
330 
Oxyhydroparacoumaric  acid,  511 
Oxymandelic  acid,  511,  513 
( bcynaphthalene,  542 
Oxyphenyl-acetic  acid,  72,  334,  511,  545 
Oxyphenvlaminopropionic     acid.     See 

Ty  rosin. 
Oxyphenylethylamine,  21,  41 
Oxyphenylpropionic   acid,    22,   334,   511, 

545 
Oxyproteic  acid  in  urine,  523.  524 
Oxyprotosulphonic  acid,  22,  23 
Oxypyrrolidincarbonic  acid,  21,  24,  80 
Oxyquinoline,  546 
Oxyquinolincarbonic  acid,  511 
Ozone,  3 
Ozone  transmitter,  169 

Palmitic  acid,  111 
Palmitin,  111 
Pancreas,  319,  320 

,  relation  to  glycolysis,  155,  258, 

259,  321 
,  extirpation  of.  action  on  absorp- 
tion, 349,  355 
,  extirpation    of,    elimination    of 

sugar,  257,  258 
,  charge  of.  323 

,  change   during   secretion,    319. 
331 
Pancreatic  calculi,  331 
Pancreatic  diabetes,  257 


694 


INDEX. 


Pancreatic  diastase,  324 
Pancreatic  proteid,  125,  320 
Pancreatic  rennin,  330 
Pancreatic  casein,  330 
Pancreatic  juice,  320 

,  secretion  of,  321,  322 
,  enzymes  of,  321,  322 
,  action      on      foodstuffs, 

324—331,  351,  354 
,  action  upon  proteoses,  43 
Parabamic  acid,  132,  484 
Paracasein,  442 
Parachymosin,  306 
Paracresol,  formation  in  putrefaction,  334, 

504 
Paraglobulin.     See  Serglobulin. 
Paraglycocholic  acid,  235 
Parahsemoglobin,  168 
Paralactic  acid,  388 

,  relation  to  formation  of 

uric  acid,  488 
,  properties  and  occurrence, 

388 
,  formation  from  glycogen, 

389,  394 
,  formation  in  osteomalacia 

bones,  369 
,  formation  in  muscle  dur- 
ing work,  396,  399 
,  formation  in  rigor  mortis, 

394 
,  formation  in  lack  of  oxy- 
gen, 389,  396,  521 
,  formation       in      animals 
with  extirpated  livers, 
389,  521 
,  passage  of,  into  the  urine, 
397,  488,  521 
Paralbumin,  235,  424 
Paralytic  saliva,  287 
Paraminophenol,  542 
Paramucin,  424,  425 
Paramyosinogen,  379,  382 
Paranuclein.     See  Pseudonuclein. 
Paranucleic  acid,  443 
Paraoxyphenylacetic  acid,  72,  334,  511 
Paraoxyphenylpropionic  acid,  22,  511 
Paraoxypropiophenone,  behavior  in   ani- 
mal body,  545 
Parapeptone,  303 
Paraxanthine,  131,  495 

in  urine,  494,  495 
Parietal  or  delomorphic  cells,  295,  307 
Parotid,  286 
Parotid  saliva,  288 
Parovarial  cysts,  422 
Pemphigus  chronicus,  224 
Penicillum  glaucum,  69 
Pentacrinin,  593 

Pentamethylendiamine.     See  Cadaverin. 
Pentosanes,  89 
Pentoses,  89 

,  relation  to  glycogen  formation, 
89,  247 
in  urine,  89,  571 


Pentoses  in  pancreas,  89 

in  nucleic  acids,  127 
in  nucleoproteids,   56,   89,   241, 
437 
Penzoldt,  acetone  reaction,  576 
Pepsin,  299—304 

,  preparation  of,  300 
,  properties,  300 

,  detection  in  gastric  contents,  313 
,  quantitative  estimation,  301,  302 
,  occurrence  in  the  urine,  356,  525 
,  occurrence  in  muscles,  383 
,  action  on  proteid,  300 
,  action  on  other  bodies,  304 
Pepsin  digestion,  300—304 

,  products  of,  41,  42,  303 
Pepsin  glands,  295,  305,  307 
Pepsin  glutin  peptone,  45 
Pepsin-hydrochloric  acid,  304 
Pepsin-like  enzyme,  299 
Pepsinogen,  295 
Pepsin  peptones,  44 
Pepsin  test,  301 
Peptides,  25,  330 
Peptochondrin,  362 
Peptones,  20,  21,  26,  29,  37—47 
in  putrefaction,  21,  334 
in    pepsin    digestion,    37 — 47, 

303, 
in  trypsin  digestion,  37 — 47 
,  assimilation  of,  344—349 
,  preparation,  46 
,  absorption  of,  344,  345 
,  passage  of,  into  urine,  346,  551 
,  action  on  the  secretion  of  gas- 
tric juice,  297 
Peptone-plasma,  143,  191 

,  carbon-dioxide     tension, 
611     . 
Peptonuria,  551 
Peptozym,  197 
Percaglobulin,  433 
Pericardial  fluid,  218,  220 
Perilymph,  418 
Peritoneal  fluid,  218,  221 
Permeability  of  the  blood-corpuscles,  164 
Peroxides  in  the  cells,  6 

,  decomposition  by  catalyses,  6,  7 
Peroxidases,  8 
Peroxyproteic  acid,  23,  524 
Perspiratio  insensibilis,  618 
Perspiration,  594 — 596 
Pettenkofer's  test  for  bile-acids,  112,  263, 
558 
respiration  apparatus,  613 
Phacozymase,  417 
Phaseomannite,  386 
Phenaceturic  acid,  503,  543 
Phenol-glucuronic  acid,  505,  546 
Phenol-sulphuric  acid  in  the  urine,  504 — 
506 
in  sweat,  596 
Phenols,  elimination   by   the   urine,   334, 
503—510,  542,  545 
in  starvation,  337 


INDEX. 


695 


Phenols,  estimation  in  the  urine,  505,  509 
,  action  on  the  urine,  506,  547 
,  formation    in    putrefaction,    21, 

334,  503,  .504 
,  behavior  in  the  animal  body,  334, 
503,  .504,  545 
Phenylacetic  acid,  formation  in  putrefac- 
tion, 21,  334 
,    behavior   in   the   ani- 
mal body,  503,  543 
Phenylalanin,  25,  41,  5\  61,  7-i 
I'heiivlaminoacetic  acid,  behavior  in  the 

animal  body,  543 
Phenylaminopropionic  acid,  21,  24 
Phenylaminopropionic    acid,  behavior   in 

the  animal  Dody,  542,  543 
Phenylglucosazone,  86,  95 
Phenylhydrazine  test,  86,  95 

in  the  urine,  563 
Phenyllactosazone,  445 
Phenylpropionic    acid,    behavior    in    the 

animal  body,  501,  543 
Phenylpropionic  acid,  formation    in    pu- 
trefaction, 21,  334,  501 
Phlebin,  165 
Phlorhizin,  241,  255,  575 

,  poisoning  with,  508 
Phlorhizin  diabetes,  255,  522 
Phloroglucin  as  reagent,  90,  314,  572 
Phosphate  calculi,  5^5 
Phosphates  in  urine,  463,  529—532,  5S1 
— 534 
.  See  also  the  different  phos- 
phates. 
Phosphatides  in  bile,  274 

in  the  brain,  407 
Phosphaturia,  530 
Phosphocarnic  acid,  3^3,  3S5 

in  the  milk,  444 
in  blood,  156 
in  brain,  408 
in  the  urine,  525 
in    relation    to    the 
elimination  of  C02 
and  lactic  acid,  394 
in  relation  to  muscu- 
lar   activity,    397, 
401 
Phosphoglucoproteid,  50,  56,  433 
Phosphoric  acid,  elimination  by  the  urine, 
529—532,  535 
,  formation     in    muscular 

activity,  397 
,  physiological  importance, 

140 
.quantitative     estimation 
of,  531 
Phosphorized  combinations  in  the  urine, 

525 
Phosphorus  poisoning,  action  on  the  elim- 
ination of  am- 
monia, 472 
,  action  on  the  elim- 
ination of  urea, 
472 


Phosphorus  poisoning,  action  on  the  elim- 
ination  of  lactic 
acid,  389,  521 
,  fatty  degeneration 

caused    by,    241, 

372 

,  liver  autolysis    in, 

VI 
,  change      in      the 
urine,  389,   472, 
580 
Photomethcemoglobin,  172 
Phrenin,  412 
Phrenosin,  410 

Phthalic  acid,  behavior  in  the  body,  542 
Phyllocyanin,  180 
Phylloporphyrin,  165,  180 
Phymatorusin,  591 

in  the  urine,  557 
Physetoelic  acid,  115 
Physiological  availability,  527 
Phytosterines,  283 

a-Picolin,  behavior  in  the  animal  body,  544 
Picric  acid,  reagent  for  proteid,  30,  553 
,  reagent  for  creatinine,  483 
,  reagent  for  sugar,  95,  565 
Pigment  calculi,  282 
Pigments  of  the  eye,  414 — 416 

of  the  blood,  164—183 
of  the  blood-serum,  156,  428 
of  the  corpora  lutea,  181,  422 
of  the  egg-shell,  432 
of  feathers,  592 
of  the  fat-cells,  370 
of  the  bile,  269—274,  276 
of  the  urine,  514—520 
of  the  skin,  590—593 
of  the  lobster,  433,  592 
of  the  liver,  241 
of  the  muscles,  382,  383 
of  lower  animals,  184,  592 
,  medicinal  pigments  in  the  urine, 
547,  560 
Pigmentary  alcoholia,  277 
Pig's  milk,  450 
Pike,  flesh  of,  403 

Pilocarpin,  action  on  the  secretion  of  in- 
testinal juice,  317 
,  action  on  the  elimination  of 

C02  in  the  stomach,  309 
,  action    on    the    secretion    of 

sweat,  596 
,  action    on    the    secretion    of 

saliva,  293 
,  action   on  the  elimination  of 
uric  acid,  486 
Piperazin  solvent  for  uric  acid,  491 
Piperidin,  491 
Piqure,  256 
Piria's  tyrosin  test,  73 
Placenta,  435 
Plant  nucleic  acids,  129 
Plants,  chemical  processes  in  the  same,  1, 2 
Plasma.     See  Blood-plasma. 
Plasminic  acid,  130 


696 


INDEX. 


Plasmoschisis,  191 

Plasmozym,  195 

Plastein,  44,  306 

Plastin,  124,  126 

Plastinogen,  44 

Plattner's  crystallized  bile,  263 

Plethora  polycythemia,  207 

Pleural  fluid,  218,  221 

Plums,  action  on  the  elimination  of  hip- 

puric  acid,  500 
Pneumonic  infiltration,  solution  of,  12,  614 
Poikilocytosis,  208 
Polarization  test,  564 
Polycythemia,  207,  210 
Polypeptides,  25 

in  tryptic  digestion,  42,  330 
Polyperythrin,  593 
Polysaccharides,  103 — 107 
Polyuria,  537 
Pons  varolii,  412 
Pork-fat,  absorption  of,  355 
Portal  vein,  blood  of,  203,  252,  349 
Positive  phase,  196 

Potassium  combinations,  division   of,    in 
the    form-ele- 
ments       and 
fluids,  139 
,  elimination     of, 
in  fevers,  533 
,  elimination     of, 
in  starvation, 
533,  631 
,  elimination     by 
the  saliva,  293 
in  the  urine,  533 
Potassium  chlorate,  poisoning  with,  171 
Potassium  phosphate  in  yolk  of  eggs,  429 
in  muscles,  391,  404 
in  cells,  139,  140 
in  spermatozoa,  421 
Potassium  sulphocyanide  in  the  urine,  523 
in    saliva,    288, 

290 
in    gastric    con- 
tents, 299 
Potatoes,  absorption  of,  in  the  intestine, 

351 
Potential  energy  of  various  foods,  624 — 

628 
Precipitins,  156 
Preglobulin,  118,  192,  231 
Preputial  secretion,  594 
Primary  proteoses,  40 
Prisoners,  food-ration  for,  664 
Proliferous  cysts,  422 
Propepsin,  307 
Propeptones,  37 

Propylamin,  solvent  for  uric  acid,  491 
Propyl  benzene,  542 
Propylene  glycol,  relation  to  formation  of 

glycogen,  248 
Prosecretin,  319,  322 
Prostatic  calculi,  422 

secretion,  419 
Prostetic  group,  56 


Protagon,  123,  231,  407,  408,  409 
Protalbinic  acid,  37 
Protoproteoses,  22,  39 
Protamins,  19,  26,  47,  80,  124,  421 
Proteid,  separation  from  fluids,  33 

,  approximate    estimation    in    the 

urine,  553 
,  circulating    and    tissue    proteid, 

641,  642 
,  action  on  the  formation  of  gly- 
cogen, 248,  249 
,  active,  4 

,  living  and  non-living,  4 
,  detection  of,  29—32 
,  detection  of,  in  urine,  547 — 550 
,  quantitative  estimation  of,  32 
,  quantitative    estimation    of,    in 

urine,  552 
,  quantitative    estimation     of,    in 

milk,  446,  447 
,  absorption  of,  344 — 349 
,  passage  of,  into  the  urine,  547 
,  heat  of  combustion  of,  626,  628 
,  digestibility  in  gastric  juice,  300, 

301,  302,  309,  310 
,  digestibility  in  pancreatic  juice, 
327,328 
Proteid  bodies  in  general,  18 — 47 

,  summary  of  the  various, 

26,  33—47 
.  See  also  the  various  pro- 
teid bodies  of  the  tissues 
and  fluids. 
Proteid  glands,  56,  2S6 
Proteid  metabolism   in  work    and    rest, 
397—401,  655 
in  starvation,  629 
in  various  ages,  654 
with  different  foods, 

639—650 
after     feeding     with 
thyroid     extracts, 
235 
Proteid   putrefaction,    16,    21,   333—340, 

501,  503,  510 
Protein,  relation  to  the  albuminates,  36 
Proteinochromogen,  21,  81 
Protein  substances,  18 — 66.     See  also  in- 
dividual protein  bodies. 
Proteoses,  26,  39,  37 

,  general   properties  and  prepa- 
ration, 37—47 
in  blood,  153,  347 
,  formation   in  proteid  putrefac- 
tion, 334 
,  formation  in   peptic  digestion, 

303 
,  relationship  to  the  coagulation 

of  the  blood,  143,  189,  196 
,  nutritive  value,  644 
,  absorption  of,  345 
,  transformation     into     proteid, 

347 
,  occurrence  in  urine,  551 
Prothrombin,  147,  192,  193,  194,  228 


INDEX. 


607 


Protic  acid,  383 

Protocatechuic  acid,  behavior  in  the  body, 
506 

Protoelastose,  60 
Protogelatose,  63 
Protogen,  37 
Protones,48 

Protoplasm,  3,  117,  118 
Protosyntonose,  80 
Pseudochylous  fluid,  222 
Pseudoglobulin,  149 
Pseudoglycogen  formers,  250 
Pseudohsemoglobin,  170 
Pseudohevulose,  N7 
Pseudomucin,  53,  423 

in  ascitic  fluids,  219 
in  cysts,  422,  423 
in  the  gall-bladder,  278 
Pseudonucleins,  34,  126,  303 

from  casein,  442,  451 
from  vitelin,  427 
,  consummation    and    ab- 
sorption, 231,  620 
Pseudopepsin,  299 
Pseudotagatose,  87 
Pseudoxanthine,  386 
Psittacofulvin,  592 
Psyllic  acid,  594 

Psychical  period  of  secretion,  296 
Psyllosteryl  alcohol,  594 
Ptomaines,  16,  21 

in  the  urine,  526,  5S0 
Ptyalin,  290 

,  behavior  with  hydrochloric  acid, 

291,  292,  331 
,  action  on  starch,  291 — 294 
Pulmotartaric  acid,  614 
Purin,  130 
Purin  bases,  130—137,  494 

,  quantitative   determination, 
137.    See  also  Nuclein  bases. 
Purple,  593 
Purple  cruorin,  170 
Pus,  226—229 
,  blue,  228 
in  urine.  558 
corpuscles,  227 
serum,  226 
Putrefactive  processes,  4,  16,  21 

in  intestine,  333 — 
340,  504—510 
Putrescin,  16 

in  intestine,  580 
in  the  urine,  526,  5S0 
Pvin,  221.  226,  228 
PyiniC  acid,  228 
Pyloric  gland,  295 
Pyloric  secretion,  308 
Pyocyanin,  229 

in  sweat,  596 
Pvogenin,  227,  409 
PVosin.  227,  409 
Pyoxanthose,  229 

Pyridine,  behavior  in  the  body,  546 
a-Pyridine-carbonic  acid,  543,  544 


a-Pyridine-uric  acid,  543 

Pyrocatechin,  506 

,  occurrence  in  urine,  506 
,  occurrence   in  transudates, 
220 
Pyrocatechin-sulphuric  acid,  506 
Pyromucic  acid,  545 
Pyromucin-ornithuric  acid,  545 
Pyrrol  derivatives,  80—82 
Pyrrolidin  carbonic  acid,  21,  24,  41,  58, 

62,80 
Pyrrolidon  carbonic  acid,  58 

Quercite,  relation  to  glycogen  formation, 

247 
Quinic  acid,  behavior  in  the  animal  body, 

500 
Quinine,  passage  of,  into  urine,  547 
,  passage  of,  into  sweat,  596 
,  action  of,  on  the  elimination  of 

uric  acid,  486 
,  action  on  the  spleen,  234 
Quotient,  respiratory,  255,  375,  399,  622, 
656 

Racemic  acid,  behavior  in  the  animal  body, 

539 
Rachitis,  bones  in,  369 
Reductases,  13 
Reduction  processes,  2,  4,  9 
Reichert's-Meissl's  equivalent,  114 
Reindeer,  milk  of,  450 
Rennin,  13,  17,  155,  304,  442 

,  detection  of,  in  gastric  contents, 

313 
,  occurrence   of,   in   the  pancreas, 

330 
,  passage  of,  into  urine,  534 
Rennin  cells,  295 
Rennin  glands,  295 
Rennin  zymogen,  295,  305 
Resacetophonon,  545 
Resin  acids,  transition  into  the  urine,  347, 

549 
Respiration,  anaerobic,  9 

,  external  598,  604 
,  internal,  598,  604,  612 
of  the  hen's  egg,  433 
of  plants,  2 
.     See  also  Chemistry  of  res- 
piration,   598 — 614,   and 
Exchange   of   gas   under 
various  conditions. 
Respiratory  quotient,  255,  375,  399,  622, 

656 
Rest,  metabolism  during,  394 — 399,  655 — 

657 
Reticulin,  26,  64,  358 
Retina,  111 
Reversion,  101 
Revertose,  14 

Reynolds'  acetone  reaction,  575 
Rhamnose,  relation    to    glycogen    forma- 
tion, 247 
Rheometer,  608 


698 


INDEX, 


Rhodizonic  acid,  387 

Rhodophan,  416 

Rhodopsin,  414 

Rhubarb,  action  on  the  urine,  547,  560 

Rib-cartilage,  363 

Rigor  mortis  of  the  muscles,  393,  394 

Roberts'    method    of    estimating    sugar, 

569 
Roch's  reaction  for  proteid,  550 
Rodents,  bile-acids  of,  265,  276 
Rods  of  the  retina,  pigments  of,  414 
Rosenbach's  bile-pigment  test,  559 

urine  test,  579 
Rotation,  specific,  88 
Rosin's  lgevulose  reaction,  97 
Rovida's  hyaline  substance,  118,  186,  227, 

419 
Rubner's  sugar  test,  95,  564 
Rye  bread  in  the  intestine,  348,  351 

Saccharic  acid,  84,  102 

,  lactone  of,  99 
,  relation  to  glycogen  forma- 
tion, 248 
Saccharose,  101 

calorific  value,  625,  626 
absorption  of,  350 
Salicylase  or  aldehydase,  8 
Salicylic  acid,  action  on  pepsin   digestion, 
303 
,  action  on  metabolism,  579 
,  action  on  trypsin  digestion, 

329 
,  behavior     in     the     animal 
body,  543 
Salicylsulphonic  acid  as  proteid  reagent, 

30 
Saliva,  286—295 

,  secretion  of,  294 
,  mixed,  289 

,  physiological  importance,  294 
,  behavior  in  the  stomach,  294 
,  action  of,  294 
,  gases  of,  603 
,  composition  of,  293 
Salivary  calculi,  294 
Salivary  diastase.     See  Ptyalin. 
Salivary  glands,  286 
Salkowski's  cholesterin  reaction,  284 
Salkowski-Ludwig's  method  of  estimating 

uric  acid,  492 
Salmin,  47 
Salmon,  flesh  of,  403 

,  sperma  of,  47,  421 
Salmonucieic  acid,  128 
Salts.     See  the  various  salts. 
Salt-plasma,  143 
Salts  of  vegetable  acids,  behavior  in  the 

organism,  464 
S; i mandarin,  594 

Santonin,  action  on  the  urine,  547,  560 
Saponification  equivalent,  114 
S.i )  lonification  of  nentral  fats,  1 10, 326, 332, 

352 
Sarcolactic  acid.     See  Paralactic  acid. 


Sarcolemma,  376 
Sarcomelanin,  591 
Sarcomelanic  acid,  591 
Sarcosin,  384 

,  behavior  in  the  animal  body,  540 
Sarkine.     See  Hypoxanthine. 
Scherer's  inosit  test,  3S7 
Schiff's  reaction  for  cholesterin,  284 
reaction  for  uric  acid,  492 
reaction  for  urea,  473 
Schreiner's  base,  420 
Schweitzer's  reagent,  107 
Sclerotica,  418 
Scombrin,  47 
Scombron,  49 
Scyllit,  232 
Scymnol,  262 

Scymnol-sulphuric  acid,  262 
Sea-urchin,  sperm  of,  49 
Sebacic  acid,  112 
Sebum,  593 

Secondary  proteoses,  40 
Secretin,  322 

Sediments.     See  Urinary  sediments. 
Sedimentum  lateritium,  463,  520,  581 
Seliwanoff's  reaction  for  lsevulose,  97,  570 
Semen,  419 

Semicarbazide,  poisoning  with,  499 
Semiglutin,  63 
Seminose.     See  Mannose. 
Senna,  action  on  the  urine,  547 
Seralbumin,  20,  23,  26,  151 

,  detection    of,    in    the    urine, 

548—550 
,  quantitative    estimation    of, 

153,  552 
,  absorption  of,  345 
Serglobulin,  20,  23,  26,  149 

,  relation  to  the  coagulation  of 

the  blood,  186,  192 
,  carbohydrate  group  of,  150 
,  detection  of,  in  the  urine,  550 
,  quantitative    estimation    of, 
153,  552 
Sericin,  26,  66,  67 
Serin,  21,  24,  58,  67 
Serosamucin,  219 
Serous  fluids,  217—226 
Serum.     See  Blood-serum. 
Serum  casein.     See  Serglobulin. 
Sex,  influence  on  metabolism,  653 
Sharks,  bile  of,  262 
,  urea  in,  467 
Sheep's  milk,  450 
Shell-membrane  of  the  hen's  egg,  57,  74, 

432 
Sheep,  gastric  juice,  299 
Silicic  acid  in  feathers,  588 
in  hair,  588 
in  urine,  536 

in  hen's  egg,  429,  432,  433 
Silicic  acid  ester  in  feathers,  588 
Silk  gelatine,  65 
Sinistrin,  animal,  55 
Silver,  in  blood,  201 


INDEX. 


699 


Skatol,  21,  60,  334,  3 

,  formation     in     putrefaction,    21, 

334,  503,  510 
,  behavior  in  the  animal  body  334, 
503,  Sip,  546 
Skatolacetic  acid,  -2 
Skatolaminoaootic  acid,  -'2,  81 
Skatolcarbonk  acid,  21,  510 
Skatol-pigment,  510 
Skatosin,  82 
Skatoxyl,  334,  510,  542 
Skatoxvlglucuronic  acid,  509,  546 
Skatoxylsulphuricacid,  507,  509 

in  sweat,  596 
Skeletins,  65 

Skeleton  at  various  ages,  367 
Skin,  588—597 

,  excretion  through,  594,  596,  618 
Sleep,  metabolism,  657 
Smegma  prseputii,  594 
Smith's  reaction  for  bile-pigments,  560 
Snail  mucin,  52 
Snake  poison,  17 

,  relation  to  the  coagulation 
of  blood,  189,  196 
Soaps  in  blood-serum,  154 
in  chyle,  213,  352 
in  pus,  227 

in  excrements,  341,  342 
in  bile,  262,  274 
,  importance  of,  in  the  emulsification 
of  fats,  326,  332,  353 
Sodium    alcoholate    as    a    saponification 

agent,  113,  572 
Sodium  chloride,  elimination  by  the  urine, 

526,  527 

,  elimination  by  the  sweat, 
596 

,  physiological  import- 
ance, 635 

,  quantitative  estimation, 

527,  528 

,  influence  on  the  quan- 
tity of  urine,  651 
,  influence  on  the  elimina- 
tion of  urea,  651 
,  influence  on  the  secretion 
of  gastric  j  uice,  307 ,635 
,  behavior  with  food  rich 

in  potassium,  635 
,  insufficient     supply     of, 

307,  635 
,  action  on  pepsin  diges- 
tion, 303 
,  action  on  trypsin  diges- 
tion, 328 
Sodium  compounds,  elimination    by    the 
urine,  533 
,  division    among    the 
form-elements  and 
fluids,  139 
.     See  also  the  various 
tissues  and  fluids. 
Sodium  phosphate  in  the  urine,  463,  529, 
530,  581 


Sodium  salicylate,  action  on  the  secretion 

of  bile,  261 
Sodium    tartrate,    relation    to    glycogen 

formation,  248 
Soldiers,  diet  of,  663,  (if,  1 
Sorbinose,  91,  97 
Sorbite,  85 

Source  of  muscular  energy,  400,  401 
Sparing  theory,  249 
Specific  rotation,  87 
Spectrophotometry,  182,  183 
Sperma,  47,  419 — 122 
Spermaceti,  115 
Spermaceti  oil,  115 
Spermatin,  421 
Spermatocele  fluids,  223 
Spermatozoa,  420,  421 
Spermin,  420 
Spermin  crystals,  420 
Spherules,  27,  426,  433 
Sphygmogenin,  237 
Spider  excrement,  guanin  therein,  133 
Spider  poison,  17 
Spiegler's  reagent,  549 
Spirographin,  53 
Spirogyra,  92,  140 
Spleen,  232,  233 

,  relation  to  formation  of  blood,  233 
,  relation  to  formation  of  uric  acid, 

234,  489 
,  relation  to  digestion,  323 
,  blood  of  the,  204 
Spleen  pulp,  232,  234 
Splitting  processes  in  general,  1,  2,  9.    See 

also  the  various  enzymes. 
Spongin,  26,  65,  66 
Sputum,  615 
Sputum  mucin,  52 
Starch,  103 
Starch,  calorific  value  of,  625 

,  hydrolytic  cleavage  by  intestinal 

juice,  312 
,  hydrolytic  cleavage  by  pancreatic 

juice,  325 
,  hydrolytic  cleavage  by  saliva,  290 
Starch  cellulose,  104 
Starch  granulose,  104 
Starvation,  action  on  the  blood,  206,  632 
,  action  on  the  urine,  337,  467, 

501,  507 
,  action  on  the  elimination  of 

indican,  337,  507 
,  action   on  the    elimination  of 

oxalic  acid,  498 
,  action  on  the  secretion  of  bile, 

260 
,  action    on    the    secretion    of 

pancreatic  juice,  321 
,  action  on  the  elimination  of 

phenol,  337 
,  action     on     metabolism,  621, 

622,  628—633 
,  death  from,  628 
Starvation  cures,  665,  666 
Steapsin,  325 


700 


INDEX. 


Stearic  acid,  110 
Stearin,  110 

,  absorption  of,  354 
Stentorin,  blue,  593 
Stercoblin,  270,  342,  514 
Stercorin,  284 
Stethal,  115 

Stokes's  reduction  fluid,  170 
Stokvis'  reaction  for  bile- pigments,  560 
Stomach,  importance  in  digestion,  310, 311 
,  relation  to  intestinal  putrefac- 
tion, 311,  340 
,  auto-digestion  of,  312 
,  digestion  in  the,  308 — 313 
Stomachic  glands,  295 
Streptococcus,  behavior  with  gastric  juice, 

312 
Stroma  fibrin,  163 
Stroma  of  the  blood-corpuscles,  163 

of  the  muscles,  380 
Strontium  salts  and  blood  coagulation,  143 
Struma  cystica,  235 

Strychnine,  passage  of,  into  the  urine,  547 
Sturgeon,  sperma  of,  47 
Sturin,  47,  48,  80 
Sublingual  glands,  286 
Sublingual  saliva,  288 
Submaxillary  glands,  286 
Submaxillary  mucin,  52,  53 
Submaxillary  saliva,  287 
Succinic  acid  in  putrefaction,  21 

in  the  fermentation  of  milk, 

438 
in  the  intestine,  334 
in  the  spleen,  232 
in  transudates,  220,  223 
in  the  thyroid  glands,  234 
in  the  animal  body,  539 
j  passage  of,  into  the  urine,  521 
,  passage  of , into  the  sweat,596 
Sugar,  relation  to  work,  396,  401 

,  formation  from  fats,  253,  401 
,  formation  from  peptones,  253 
Sugar  formation,  in  the  liver,  250 — 259 

after  pancreas  extirpa- 
tion, 258 
Sugar,  behavior  on  subcutaneous  injection, 
250 
,  behavior  to  blood-corpuscles,  188 
.      See  also  various  kinds  of  sugar. 
Sugar  tests  in  the  urine,  501 — 570 
Sulphsemoglobin,  174 
Sulphocyanides  in  the  urine,  523 

in  the  saliva,  288,  290 
Sulphonal  intoxication,  urine  in,  179,   556 
Snl phonic  acids,  behavior  in  the  animal 

body, 540 
Sulphur,   of  proteids,    18.    20.     See  also 
various  proteids. 
in  the  urine,  398,  523 
,  elimination  of,  in  activity,  398 
,  elimination  of,  with  lack  of  oxy- 
gen, 523 
,  neutral    and    acid    sulphur    in 
urine,  523 


Sulphur,  behavior  in  the  organism,  523, 

540 
Sulphur  methsemoglobin,  174 
Sulphuretted  hydrogen  in  putrefaction  in 
the  intestine,  334, 
336 
in  the  urine,  524 
Sulphuric  acid,  ethereal,  and  sulphate  in 
the  urine,  503,  504,  532 
,  elimination  of,  in  activity, 

398 
,  elimination     of,     by     the 

urine,  463,  532 
,  elimination     of,     by    the 

sweat,  596 
,  estimation  of,  532 
,  relation  to  elimination  of 

nitrogen,  398,  523,  532 
,  action  on  pepsin  digestion, 
302 
Suprarenal  capsule,  236,  237,  278 
Suprarenin,  237.     See  also  Adrenalin. 
Sweat,  594—597 

Swimming-bladders  of  fishes,  gases  of,  613 
,  guanine    in, 
133 
Sympathetic  saliva,  287 
Synproteose,  43 
Synovial  fluid,  225 
Synovin,  225 
Synthesis,  1,  2 

of  ethereal  sulphuric  acids,  334, 

503,  507,  510,  545 
of  proteid,  25 
of  conjugated  glucuronic  acids, 

505,  509,  522,  541,  546 
of  uric  acid,  4S4,  488 
of  urea,  467,  470,  471 
of  hippuric  acid,  2,  14,  500 
of  varieties  of  sugars,  85,  92 
of  polypeptides,  25 
Syntonin,  36,  80,  380 

,  calorific  value  of,  625,  626 

Tagatose,  87 

Talonic  acid,  98 

Talose,  87,  98 

Tapeworm  cysts,  225 

Tannic  acid,  behavior  in  the  animal  body, 

545 
Tartar,  294 

Tartaric  acid,  relation  to  glycogen  forma- 
tion, 248 
,  passage  of,  into  sweat,  596 
behavior  in  the  animal  body, 
539 
Tatalbumin,  429 
Taurin,  76,  77,  265,  280 

,  behavior  in  the  animal  body,  540 
Taurocarbamic  acid,  540 
Taurocholic  acid,  262,  265 

,  quantity  in  various  biles, 

276 
,  occurrence  in  meconium, 
343 


INDEX. 


701 


Taurocholic  acid,  decomposition  in  the  in- 
testine, 337 
,  proteid-precipitating  ac- 
tion, 30,  554 
Tea,  action  on  metabolism,  652 
Tears,  lis 
Teeth,  369 

Teichmann's  crystals,  178,  556 
Tiinlon  mucin,  52 
Tendon  mucoid,  35S 
Tendon  synovia,  225 

Tension  of  the  C02  in  the  blood,  610 — 613 
in  the  tissues,  612 
in  the  lymph,  212 
O  in    the    blood,    604 — 
610 
Terpen-glucuronic  acid,  546 
Terpentine,  action  of,  on  the  secretion  of 
bile,  261 
,  action  of,  on  the  urine,  547 
,  behavior  in  the  animal  body, 
522,  546 
Tetronerythrin,  184,  592 
Testis,  419 
Tetroses,  84 
Theobromine,  131 

,  behavior     in     the     animal 
body,  494 
Theophylline,  131 

,  behavior  in  the  animal  body, 
494 
Thioalcohols,  behavior  in  the  animal  body, 

540 
Thioglycolic  acid,  58 

,  behavior  in  the  animal 
body,  540 
Thiolactic  acid,  20,  24,  58,  76 
Thiophene,  645 
Thiophenic  acid,  545 
Thiophenuric  acid,  545 
Thrombin,  13,  146,  147,  192—196,  228 
Thrombosin,  193 
Thymin,  127,  138 
Thymic  acid,  128 
Thvmonucleic  acid,  127,  128,  129 
Thymus,  229 
Thyreoglobulin,  234,  236 
Thyreoproteid,  236 
Thyreotozalbumin,  236 
Thyroid  gland,  234—236 
Thyroiodm.     See  Iodothyrin. 
TiflBue-fibrinogen,  118,  231 
Tissue  proteids,  641,  642 
Tollen's  reaction  for  pentoses,  90 
Toluene,   behavior  in   the  animal   body, 

500,  542 
Toluric  acid,  543 

Toluylendiamine,  poisoning  with,  281 
ToluiC  acid,  513 

Tonus,  chemical  of  the  muscle,  395 
Tooth  tissue,  369 
Tortoise,  bones  of,  366 
Tortoise-shell,  57,  593 
Toxalbumins,  behavior  with  gastric  juice, 
312 


Toxins  17,  166,  239 
Tracheal  cartilage,  58 

Transudates,  217—226,  604 
Tribromacetic  acid,  23 
Tribromaniinobenzoic  acid,  23 
Tricalcium  casein,  441 
Trichloracetic  acid  as  reagent,  30,  33 
TricUorethyl-glucuronic  acid.     See  Uro- 

chloralic  acid. 
Triolein,  111 
Trioses,  84 
Tripalmitin,  111 

Triple  phosphate  in     urinary    sediments, 
681,  5S3 
in  urinary  calculi,  584, 
5S5 
Tristearin,  110 
Triticonueleic  acid,  127,  129 
Trommer's  test  for  sugar,  94,  561 

,  behavior     with 

uric  acid,  401 
,  behavior      with 
creatinine,  482 
Tropics,  metabolism  in  inhabitants  of,  659 
Trypsin,  155,  326,  327 

,  action  on  proteids,  327 
,  action  on  other  substances,  329 
Trypsin  digestion,  327 

,  products  of,  329 
,  action  on  peptides,  330, 
348 
Trypsin  peptone,  41 
Trypsin  zymogen,  321,  327 
Tryptophan,  21,  24,  41,  82,  299 
Tubo-ovarial  cysts,  435 
Tunicin,  589 
Turacin,  592 
Turacoverdin,  592 
Tyrosin,  24,  43,  48,  58,  72,  73 
,  in  urine,  579 
,  in  sediments,  583 
,  detection  of,  73,  579 
,  origin  of,  22,  72,  334 
,  behavior    in    putrefaction,    334, 

501,  503 
,  behavior  in  the  animal  body,  512, 
542 
Tyrosinases,  8,  73,  592 
Tyrosin-sulphuric  acid,  73 

Uffelmann's  reaction  for  lactic  acid,  314 
Umikoff  s  reaction,  453 
Uracil,  127,  129,  137,  231 
Uraemia,  bile  in,  277 

,  gastric  contents  in,  313 
,  sweat  in,  596 
Uraminobenzoic  acid,  544 
Urates,  491 

,  in  sediments,  462,  580, 
Urea,  467 

,  elimination  in  starvation,  467.  633 
,  elimination  in  children,  468,  654 
,  elimination  in  disease,  468,  472,  473, 

534 
,  properties  and  reactions,  473 


702 


INDEX. 


Urea,  formation  and  origin,  469 — 473,  534 
quantitative  estimation,  475 — 480 
,  synthesis,  467,  469—472 
occurrence  in  the  blood,  156,  202, 
204,  467 
,  occurrence  in  the  bile,  262,  274,  467 
,  occurrence  in  the  liver,  467,  470 
,  occurrence  in  the  muscles,  383,  467 
,  occurrence  in  transudates,  220 
Urea  nitrate,  474 
Urea  oxalate,  474 
Ureids,  21,  484,  499 
Urein,  480 
Urethan.     See  Carbamic  acid  ethyl-ester, 

480 
Uric  acid,  131,  484 

,  elimination  in  disease,  486,  487 
,  elimination  after  feeding  with 

nuclein,  486 
,  relation  to  urea,  484,  489 
,  properties  and  reactions,  490 — 

492 
,  formation  in  the  animal  body, 

486^90 
,  quantitative  estimation,  492 — 

494 
,  syntheses  of,  489 
,  behavior  in  the  animal  body,  489 
,  occurrence  of,  485 
,  occurrence  of,  in  sweat,  596 
,  occurrence  of,  in  sediments,  462, 
491,  582 
Uric-acid  calculi,  584 
Urinary  calculi,  584 — 587 
Urinary  pigments,  514 — 521 

,  medicinal,  547,  560 
Urinary  sand,  584 
Urinary  sediments,  462,  581 — 584 
Urine,  461—587 

,  excretion  of,  536 

,  inorganic  constituents  of,  526 — 536 

,  poisonous  constituents  of,  526 

,  organic    pathological    constituents 

of,  547—580 
,  phvsiological  constituents  of,  467 — 

526 
,  enzymes  of,  525 
,  casual  constituents  of,  538 — 547 
,  color  of,  463,  514,  537,  547,  554— 

557,  558,  561 
,  solids,  calculation  of.  537 
,  quantity  of  solids,  538 
,  alkaline  fermentation  of,  581 
,  acid  fermentation  of,  581 
,  gases  of,  536 
,  quantity  of,  536—538 
,  physical  properties  of,  462 — 467 
,  osmotic  pressure  of,  465 
,  physico-chemical  analysis  of,  538 
,  reaction  of,  463 — 465 
,  acidity  of,  463,  464 
,  estimation  of  acidity,  464 
,  specific  gravity  of,  466,  537 
,  passage  of  foreign  bodies  into,  538 
—547 


Urine,  reducing  power  of,  521 
,  composition  of,  538 

Urine  indican,  507 

Urine  indigo,  507,  514 

Urine  poison,  526 

Urine  purins,  endogenous  and  exogenous, 
487 

Urine  sugar.     See  Dextrose. 

Urinometer,  466 

Urobilin,  514,  515—520 

,  relation  to  bilirubin,  270,  280, 

516 
,  relation  to  choletelin,  516 
,  relation  to  ha?matin,  280,  516 
,  relation    to    hsematoporphyrin, 

180,  516 
,  relation  to  hydrobilirubin,  280, 
517 

Urobilin  icterus,  517 

Urobilinogen,  514,  518 

Urobilinoid  bodies,  516 

Urocarnic  acid,  526 

Urochloralic  acid,  99,  541 

Urochrome,  514,  515 

Urocyanin,  514 

Uroerythrin,  514,  520 

Uroferric  acid,  525 

in  urine,  523 

Urofuscohaematin,  557 

Uroglaucin,  514 

Urohsematin,  514 

Urohodin,  514 

Uroleucic  acid,  511,  513 

Uromelanins,  514 

Uronitrotoluolic  acid,  546 

Urophsein,  514 

Uroprotic  acid,  524 

Urorubin,  514 

Urorubrohsematin,  557 

Urorosein,  514,  557 

Urospectrin,  556 

Urostealith,  5S6 

Urotheobromine,  495 

Urotoxic  coefficient,  526 

Uroxanthine,  507 

Uroxonic  acid,  485 

Ursocholeic  acid,  268 

Uterine  milk,  435 

Uterus  colloid,  425 

Utilization  of  the  various  foodstuffs,  348, 
354,  659 

Valerianic  acid,  21 

Vegetable   acids,   behavior  of  the  alkali 

salts  of,  in  body,  464 
Vegetable  gums,  105,  106 
Vegetable  mucilages,  105,  106 
Vegetarians,  food  of,  648,  661 

,  excrement  from,  341 
Vernix  caseosa,  283,  593 
Vesicatory  blisters,  224 
Vesicle  calculi,  5S4 
Vesiculasc,  420 
Visual  purple,  414,  415 
Visual  red,  414 


INDEX. 


To:; 


Vitali'a  pus-blood  test,  555 
Vit.'llin,  26 

in  yolk  of  egg,  426 

in  protoplasm,  118 
Yitrll. .lutein,  428 
Vitellorubin,  128 
Vitelloaes,  39 
Vitreous  humor,  416 

\\  ater,  drinking  of,  action  in  the  elimina- 
tion    of     chlorides, 
527 
,  action  on  the  elimina- 
tion of  uric  acid,  4S6 
,  action  on  the  elimina- 
tion of  urea,  655 
,  action  on  the  deposi- 
tion of  fat,  655 
,  action  on   the  excre- 
tion of  urine,  536 
,  elimination  of,  by  the  urine,  536 — 

538,  618 
,  elimination  of,  by  the  skin,  594,  618 
,  elimination  of,  in  starvation,  631 
,  elimination  of,  importance  for  the 

animal  body,  633 
,  elimination  of,  quantity  of,  in  the 

various  organs,  633 
,  elimination,  lack  of,  in  the  food, 
633 
Wax,  113 

in  plants,  593 
Weidel's  xanthine  reaction,  133 
Weyl's  reaction  for  creatinine,  482 
Wheat  bread,  absorption  of,  351 
Whey,  438,  449 
Whey  proteid,  442 
White  of  egg,  429 

,  calorific  value  of,  625 
,  absorption  of,  345 
Witch's  milk,  455 
Woman's  milk.     See  Human  milk. 
Wool-fat,  285,  594 

Work,  action     on     the     elimination     of 
chlorine,  527 
,  action  on  the  elimination  of  phos- 
phoric acid,  530 


Work,  action  on   the   elimination  of  sur- 
phur,  398 
,  action   on   the   excretion  of  nitro- 
gen, 398 
,  action   on  the  necessity  for  food, 

663 
,  action   on    metabolism,   394 — 402, 
655—657 
Worm-Muller's  sugar  test,  562 

Xanthine,  131,  132 

in  the  urine,  494 
in  urinary  calculi,  586 
in  urinary  sediments,  583 
,  detection  and  quantitative  esti- 
mation, 136,  137,  496 
Xanthine  bodies.      See  Nuclein  bases. 
Xanthine  calculi,  586 
Xantho-creatinine,  386,  398,  484 
Xantho-melanin,  23 
Xanthophan,  416 
Xanthoproteic  reaction,  31 
Xylene,  behavior  in  the  animal  body,  543 
Xyloses,  91,  107 

,  relation     to    the    formation    of 
glycogen,  89,  247 

Yeast-cells,  relation  to  fermentation,  10, 

11 
Yeast  maltose,  14 
Yeast  nucleic  acid,  127,  129 
Yeast  nuclein,  125 
Yolk  of  the  hen's  egg,  428,  429 
Yolk-spherules,  426,  433 

Zein,  19 

Zinc   in  the  bile,  274 
in  the  liver,  244 
,  passage  of,  into  milk,  459 
Zooerythrin,  592 
Zoofulvin,  592 
Zooid,  162 
Zoorubin,  592 
Zymase  from  beer-yeast,  11 

in  pancreas,  331 
Zymogens,  11.     See  various  enzymes. 
Zymoplastic  substances,  192,  196 


SHORT-TITLE     CATALOGUE 

OF  THE 

PUBLICATIONS 

OF 

JOHN   WILEY    &    SONS, 

New  York. 
London:   CHAPMAN  &  HALL,  Limited. 


ARRANGED  UNDER  SUBJECTS. 


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1 


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2 


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Chlorination  Process i2mo, 


I 

50 

3 

00 

3 

00 

1 

00 

2 

00 

3 

00 

3 

(JO 

I 

50 

I 

50 

2 

50 

3 

50 

4 

00 

2 

50 

3 

00 

2 

50 

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Erdmann's  Introduction  to  Chemical  Preparations.     (Dunlap.) umo,  1  25 

3 


3 

00 

3 

So 

1 

50 

2 

50 

1 

So 

1 

So 

•1 

00 

3 

00 

2 

00 

Fletcher's  Practical  Instructions  in  Quantitative  Assaying  with  the  Blowpipe. 

iamo,  morocco,  i  50 

Fowler's  Sewage  Works  Analyses i2mo,  2  o« 

Fresenius's  Manual  of  Qualitative  Chemical  Analysis.     (Wells.) 8vo,  5  o« 

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*  Martin's  Laboratory  Guide  to  Qualitative  Analysis  with  the  Blowpipe .  .  nmo,  60 
Mason's  Water-supply.     (Considered  Principally  from  a  Sanitary  Standpoint.) 

3d  Edition,  Rewritten 8vo,  4  00 

Examination  of  Water.     (Chemical  and  Bacteriological.) nrro,  1  25 

Matthew's  The  Textile  Fibres 8vo,  3  50 

Meyer's  Determination  of  Radicles  in  Carbon  Compounds.     (Tingle.),  .nmo,  1  00 

Miller's  Manual  of  Assaying nmo,  1  00 

Mixter's  Elementary  Text-book  of  Chemistry nmo,  1  50 

Morgan's  Outline  of  Theory  of  Solution  and  its  Results nmo,  1  00 

Elements  of  Physical  Chemistry nmo,  2  00 

Morse's  Calculations  used  in  Cane-sugar  Factories i6mo,  morocco,  1   50 

Mulliken's  General  Method  for  the  Identification  of  Pure  Organic  Compounds. 

Vol.  I Large  8vo,  5  00 

O'Brine's  Laboratory  Guide  in  Chemical  Analysis 8vo,  2  00 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  00 

Ostwald's  Conyersations  on  Chemistry.     Part  One.     (Ramsey.) nmo,  1  50 

Ostwald's  Conversations  on  Chemistry.     Part  Two.     (Turnbull.).     (In  Press.) 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  50 

Pictet's  The  Alkaloids  and  their  Chemical  Constitution.     (Biddle.) 8vo,  5  00 

Pinner's  Introduction  to  Organic  Chemistry.     (Austen.) nmo,  1  50 

Poole's  Calorific  Power  of  Fuels 8vo,  3  00 

Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Fefcr- 

encc  to  Sanitary  Water  Analysis nmo,  1  25 

4 


*  Reisig's  Guide  to  Piece-dyeing Svo,  25  OO 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sanitary  Standpoint  8vo,  2  00 

Richards's  Cost  of  Living  as  Modified  by  Sanitary  Science umo,  1  00 

Cost  of  Food,  a  Study  in  Dietaries 12 mo,  1  00 

*  Richards  and  Williams's  The  Dietary  Computer 8vo,  1  50 

Ricketts  and  Russell's  Skeleton  Notes  upon  Inorganic   Chemistry.      I  Part  I. 

Non-metallic  Elements.) 8vo,  morocco,  75 

Ricketts  and  Miller's  Notes  on  Assaying 8vo,  3  00 

Ridtal's  Sewage  and  the  Bacterial  Purificat  on  of  Sewage 8vo,  3  50 

Disinfection  and  the  Preservation  of  Food 8vo,  4  00 

Rigg's  Elementary  Manual  for  the  Chemical  Laboratory.  .                            Svo,  1  25 

Rostoski's  Serum  Diagnosis.     (Bolduan.l i2mo,  1  00 

Ruddiman's  Incompatibilities  in  Prescriptions 8vo,  2  00 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vo,  3  00 

Salkowski's  Physiological  and  Pathological  Chemistry.     <  Orndorff. 8vo,  2  50 

Schimpf's  Text-book  of  Volumetric  Analysis i2mo,  2  50 

Essentials  of  Volumetric  Analysis nmo,  1  25 

Spencer's  Handbook  for  Chemists  of  Beet-sugar  Houses i6mo,  morocco.  3  00 

Handbook  for  Sugar  Manufacturers  and  their  Chemists.  .  i6mo,  morocco,  2  00 

Stockbridge's  Rocks  and  Soils 8vo,  2  50 

*  Tillman's  Elementary  Lessons  in  Heat 8vo,  1  50 

*  Descriptive  General  Chemistry 8vo,  3  00 

Treadwell's  Qualitative  Analysis.     (Hall.) 8vo.  3  00 

Quantitative  Analysis.     (Hall.) 8vo,  4  00 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  00 

Van  Deventer's  Physical  Chemistry  for  Beginners.     (Boltwood.) nmo,  1  50 

*  Walke's  Lectures  on  Explosives Svo,  4  00 

Washington's  Manual  of  the  Chemical  Analysis  of  Rocks 8vo,  2  00 

Wassermann's  Immune  Sera:  Haemolyslns,  Cytotoxics,  and  Precipitins.    1B0I- 

duan.) i2mo,  1  00 

Well's  Laboratory  Guide  in  Qualitative  Chemical  Analysis 8vo,  1  50 

Short  Course  in  Inorganic  Qualitative  Chemical  Analysis  for  Engineering 

Students nmo,  1  50 

Text-book  of  Chemical  Arithmetic.     (In  press.) 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

Wilson's  Cyanide  Processes nmo,  1  50 

Chlorination  Process nmo,  1  50 

Wuiling's    Elementary    Course    in  Inorganic,  Pharmaceutical,  and  Medical 

Chemistry nmo,  2  00 

CIVIL  ENGINEERING. 
BRIDGES    AND    ROOFS.       HYDRAULICS.       MATERIALS   OF    ENGINEERING. 
RAILWAY  ENGINEERING. 

Baker's  Engineers'  Surveying  Instruments nmo,  3  00 

Bixby's  Graphical  Computing  Table Paper  10*  >  24}  inches.  25 

**  Burr's  Ancient  and  Modern  Engineering  and  the  Isthmian  Canal.     (Postage, 

27  cents  additional.) 8vo,  3  50 

Comstock's  Field  Astronomy  for  Engineers 8vo,  2  50 

Davis's  Elevation  and  Stadia  Tables 8vo,  1  00 

Elliott's  Engineering  for  Land  Drainage nmo,  1  50 

Practical  Farm  Drainage nmo,  1  00 

Fiebeger's  Treatise  on  Civil  Engineering.     (In  press.  1 

Folwell's  Sewerage.     (Designing  and  Maintenance. Svo,  3  00 

Freitap's  Architectural  Engineering.     2d  Edition,  Rewritten Svo,  3  50 

French  and  Ives's  Stereotomy »8vo,  2  50 

Goodhue's  Municipal  Improvements nmo,  1  75 

Goodrich's  Economic  Disposal  of  Towns'  Refuse 8vo,  3  50 

Gore's  Elements  of  Geodesy Svo,  2  50 

Hayford's  Text-book  of  Geodetic  Astronomy 8vo.  3  00 

Hering's  Ready  Reference  Tables  (Conversion  Factors^ i6mo,  rr.orccco  2  50 

5 


Howe's  Retaining  Walls  for  Earth i2mo,  i  25 

Johnson's  (J.  B.)  Theory  and  Practice  of  Surveying Small  8vo,  4  00 

Johnson's  (L.  J.)  Statics  by  Algebraic  and  Graphic  Methods 8vo,  2  00 

Laplace's  Philosophical  Essay  on  Probabilities.     (Truscott  and  Emory.),  nmo,  2  00 

Mahan's  Treatise  on  Civil  Engineering.     (1873.)     (Wood.) 8vo,  5  00 

*  Descriptive  Geometry 1 8vo,  1  50 

Merriman's  Elements  of  Precise  Surveying  and  Geodesy 8vo,  2  50 

Elements  of  Sanitary  Engineering 8vo,  2  00 

Merriman  and  Brooks's  Handbook  for  Surveyors i6mo,  morocco,  2  00 

Nugent's  Plane  Surveying 8vo,  3  50 

Ogden's  Sewer  Design nmo,  2  00 

Patron's  Treatise  on  Civil  Engineering 8vo  half  leather,  7  50 

Reed's  Topographical  Drawing  and  Sketching 4T0,  5  00 

Rideal's  Sewage  and  the  Bacterial  Purification  of  Sewage 8vo,  3  50 

Siebert  and  Biggin's  Modern  Stone-cutting  and  Masonry 8vo,  1  50 

Smith's  Manual  of  Topographical  Drawing.     (McMillan.) 8vo,  2  50 

Sondericker's  Graphic  Statics,  with  Applications  to  Trusses,  Beams,  and  Arches. 

8vo,  2  oo 

Taylor  and  Thompson's  Treatise  on  Concrete,  Plain  and  Reinforced 8vo,  5  00 

*  Trautwine's  Civil  Engineer's  Pocket-book i6mo,  morocco,  5  00 

Wait's  Engineering  and  Archi'ectural  Jurisprudence 8vo,  6  00 

Sheep,  6  50 
Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture  8vo,  5  00 

Sheep,  5  50 

Law  of  Contracts 8vo,  3  00 

Warren's  Stereotomy — Problems  in  Stone-cutting 8vo,  2  50 

Webb's  Problems  in  the  Use  and  Adjustment  of  Engineering  Instruments. 

i6mo,  morocco,  1   25 

*  Wheeler  s  Elementary  Course  of  Civil  Engineering 8vo,  4  00 

Wilson's  Topographic  Surveying 8vo,  3  50 

BRIDGES  AND  ROOFS. 

Boiler's  Practical  Treatise  on  the  Construction  of  Iron  Highway  Bridges.  .8vo,  2  00 

*  Thames  River  Bridge 4to,  paper,  5  00 

Burr's  Course  on  the  Stresses  in  Bridges  and  Roof  Trusses,  Arched  Ribs,  ard 

Suspension  Bridges 8vo,  3  50 

Burr  and  Falk's  Influence  Lines  for  Bridge  and  Roof  Computations.  .  .   8vo,  3  00 

Du  Bois's  Mechanics  of  Engineering.     Vol.  II Small  410,  10  00 

Foster's  Treatise  on  Wooden  Trestle  Bridges 410,  5  00 

Fowler's  Ordinary  Foundations 8vo,  3  50 

Greene's  Roof  Trusses 8vo,  1   25 

Bridge  Trusses 8vo,  2  50 

Arches  in  Wood,  Iron,  and  Stone 8vo,  2  50 

Howe's  Treatise  on  Arches 8vo,  4  00 

Design  of  Simple  Roof-trusses  in  Wood  and  Steel '.  .8vo,  2  00 

Johnson,  Bryan,  and  Turneaure's  Theory  and  Practice  in  the  Designing  of 

Modern  Framed  Structures Small  4to,  10  00 

Merriman  and  Jacoby's  Text-book  on  Roofs  and  Bridges: 

Part  I.     Stresses  in  Simple  Trusses 8vo,  2  50 

Part  II.     Graphic  Statics 8vo,  2  50 

Part  III.     Bridge  Design 8vo,  2  50 

Part  IV.     Higher  Structures 8vo,  2  50 

Morison's  Memphis  Bridge 4to,  10  00 

Waddell's  De  Pontibus,  a  Pocket-book  for  Bridge  Engineers.  .  i6mo,  morocco,  3  00 

Specifications  for  Steel  Bridges i2mo,  1   25 

Wood's  Treatise  on  the  Theory  of  the  Construction  of  Bridges  and  Roofs.  .  8vo,  2  c  ? 
Wright's  Designing  of  Draw-spans: 

Part  I.     Plate-girder  Draws 8vo,  2  50 

Part  II.     Riveted-truss  and  Pin-connected  Long-span  Draws 8vo,  2  50 

Two  parts  in  one  volume 8vo,  3  50 

6 


HYDRALLICS. 

Bazin's  Experiments  upon  the  Contraction  of  the  Liquid  Vein  Issuing  from 

an  Orifice.     (Trautwine.) 8vo,  2  00 

Bovey's  Treatise  on  Hydraulics 8vo,  5  00 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Diagrams  of  Mean  Velocity  of  Water  in  Open  Channels pa;>er,  1  50 

Coffin's  Graphical  Solution  of  Hydraulic  Problems i6mo,  morocco,  2  50 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  00 

Folwell's  Water-supply  Engineering 8vo,  4  00 

Frizell's  Water-power. 8vo,  5  c» 

Fuertes's  Water  and  Public  Health i2mo,  1  50 

Water-filtration  Works nmo,  2  50 

Ganguillet  and  Kutter's  General  Formula  for  the  Uniform  Flow  of  Water  in 

Rivers  and  Other  Channels.     (Hering  and  Trau   vine.) 8vo  4  00 

Hazen's  Filtration  of  Public  Water-supply 8vo,  3  00 

Hazlehurst's  Towers  and  Tanks  for  Water-works 8vo,  2  50 

Herschel's  115  Experiments  on  the  Carrying  Capacity  of  Large,  Riveted,  Metal 

Conduits 8vo,  2  00 

Mason's  Water-supply.     (Considered  Principally  from  a  Sanitary  Standpoint.) 

8vo,  4  00 

Merriman's  Treatise  on  Hyiraulics 8vo,  5  00 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  00 

Schuyler's   Reservoirs   for   Irrigation,   Water-power,   and   Domestic   Water- 
supply Large  8vo,  5  00 

**  Thomas  and  Watt's  Improvement  of  Rivers.     (Post.,  44c.  additional.). 4to,  6  00 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  00 

Wegmann's  Design  and  Construction  of  Dams 4to,  5  00 

Water-supply  of  the  City  of  New  York  from  1658  to  1895 4to,  10  00 

Wilson's  Irrigation  Engineering Small  8vo,  4  00 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  00 

Wood's  Turbines 8vo,  2  50 

Elements  of  Analytical  Mechanics 8vo,  3  00 

MATERIALS   OF  ENGINEERING. 

Baker's  Treatise  on  Masonry  Construction 8vo, 

Roads  and  Pavements 3vo, 

Black's  United  States  Public  Works Oblong  4to» 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8ve. 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering 8vo, 

Byrne's  Highway  Construction 8vo, 

Inspection  of  the  Materials  and  Workmanship  Employed  in  Construction. 

i6mo. 

Church's  Mechanics  of  Engineering 8vo, 

Du  Bois's  Mechanics  of  Engineering.     VoL  I Small  4to, 

Johnson's  Materials  of  Construction Large  8vo, 

Fowler's  Ordinary  Foundations 8vo, 

Keep's  Cast  Iron 8vo, 

Lanza's  Applied  Mechanics 8vo, 

Marten's  Handbook  on  Testing  Materials.     (Henning. )     2  vols 8vo, 

Merrill's  Stones  for  Building  and  Decoration 8vo, 

Merriman's  Text-book  on  the  Mechanics  of  Materials 8vo, 

Strength  of  Materials nmo, 

iietcalf's  Steel.     A  Manual  for  Steel-users nmo, 

Patton's  Practical  Treatise  on  Foundations .. 8vo, 

Richardson's  Modern  Asphalt  Pavements 8vo, 

Richey's  Handbook  for  Superintendents  of  Construction lomo,  mor., 

Rockwell's  Roads  and  Pavements  in  France 12 mo, 

7 


5 

00 

5 

00 

5 

00 

7 

50 

7 

90 

5 

'•a 

3 

00 

6 

00 

7 

90 

6 

00 

3 

50 

2 

50 

7 

50 

7 

50 

5 

00 

4 

00 

I 

00 

2 

•  •0 

5 

00 

3  00 

4 

00 

I 

25 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vo,  3  00 

Smith's  Materials  of  Machines i2mo,  1  00 

Snow's  Principal  Species  of  Wood .- 8vo,  3  50 

Spalding's  Hydraulic  Cement i2mo,  2  00 

Text-book  on  Roads  and  Pavements i2mo,  2  00 

Taylor  and  Thompson's  Treatise  on  Concrete,  Plain  and  Reinforced. 8vo,  5  00 

Thurston's  Materials  of  Engineering.     3  Parts 8vo,  8  00 

Part  I.     Non-metallic  Materials  of  Engineering  and  Metallurgy 8vo,  2  00 

Part  II.     Iron  and  Steel 8vo,  3  50 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Thurston's  Text-book  of  the  Materials  of  Construction 8vo,  5  00 

Tillson's  Street  Pavements  and  Paving  Materials 8vo,  4  00 

Waddell's  De  Pontibus.    f*  Pocket-book  for  Bridge  Engineers.).  .i6mo,  mor.,  3  00 

Specifications  for  Stt  i  Bridges i2mo,  1  25 

Wood's  (De  V.)  Treatise  on  the  Resistance  of  Materials,  and  an  Appendix  on 

the  Preservation  of  Timber 8vo,  2  00 

Wood's  (De  V.)  Elements  of  Analytical  Mechanics 8vo,  3  00 

Wood's  (M.  P.)  Rustless  Coatings:    Corrosion  and  Electrolysis  of  Iron  and 

Steel 8vo,  4  00 

RAILWAY  ENGINEERING. 

Andrew's  Handbook  for  Street  Railway  Engineers 3x5  inches,  morocco,  1  25 

Berg's  Buildings  and  Structures  of  American  Railroads 4to,  5  00 

Brook's  Handbook  of  Street  Railroad  Location i6mo,  morocco,  1  50 

Butt's  Civil  Engineer's  Field-book i6mo,  morocco,  2  50 

Crandall's  Transition  Curve i6mo,  morocco,  1  50 

Railway  and  Other  Earthwork  Tables 8vo,  1  50 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  .i6mo,  morocco,  5  00 

Dredge's  History  of  the  Pennsylvania  Railroad:    (1879) Paper,  5  00 

*  Drinker's  Tunnelling,  Explosive  Compounds,  and  Rock  Drills. 4to,  half  mor.,  25  00 

Fisher's  Table  of  Cubic  Yards Cardboard,  25 

Godwin's  Railroad  Engineers'  Field-book  and  Explorers'  Guide.  .  .  i6mo,  mor.,  2  50 

Howard's  Transition  Curve  Field-book i6mo,  morocco,  1  50 

Hudson's  Tables  for  Calculating  the  Cubic  Contents  of  Excavations  and  Em- 
bankments  8vo,  1  00 

Molitor  and  Beard's  Manual  for  Resident  Engineers i6mo,  1  00 

Wagle's  Field  Manual  for  Railroad  Engineers i6mo,  morocco,  3  00 

Philbrick's  Field  Manual  for  Engineers i6mo,  morocco,  3  00 

Searles's  Field  Engineering i6mo,  morocco,  3  00 

Railroad  Spiral i6mo,  morocco,  1  50 

Taylor's  Prismoidal  Formulae  and  Earthwork 8vo,  1  50 

*  Trautwine's  Method  of  Calculating  the  Cube  Contents  of  Excavations  and 

Embankments  by  the  Aid  of  Diagrams 8vo,  2  00 

The  Field  Practice  of  Laying  Out  Circular  Curves  for  Railroads. 

.,        i2mo,  morocco,  2  50 

Cross-section  Sheet Paper,  25 

Webb's  Railroad  Construction i6mo,  morocco,  5  00 

Wellington's  Economic  Theory  of  the  Location  of  Railways Small  8vo,  5  00 

DRAWING. 

Barr's  Kinematics  of  Machinery 8vo,  2  50 

*  Bartlett's  Mechanical  Drawing 8vo,  3  00 

*  "                                          "        Abridged  Ed 8vo,  1  50 

Coolidge's  Manual  of  Drawing 8vo,  paper  1  00 

Coolidge  and  Freeman's  Elements  of  General  Drafting  for  Mechanical  Engi- 
neers  Oblong  4to,  2  50 

Durley's  Kinematics  of  Machines 8vo,  4  00 

Emch's  Introduction  to  Projective  Geometry  and  its  Applications 8vo.  2  50 

8 


Hill's  Text-book  on  Shades  and  Shadows,  and  Perspective 8vo, 

Jamison's  Elements  of  Mechanical  Drawing 8vo, 

Jones's  Machine  Design: 

Part  I.     Kinematics  of  Machinery 8vo, 

Part  n.     Form,  Strength,  and  Proportions  of  Parts 8vo, 

MacCord's  Elemonts  of  Descriptive  Geometry 8vo, 

Kinematics;   or,  Practical  Mechanism 8vo, 

Mechanical  Drawing 4to, 

Velocity  Diagrams 8vo, 

*  Mahan's  Descriptive  Geometry  and  Stone-cutting 8vo, 

Industrial  Drawing.     (Thompson.) 8vo, 

Moyer's  Descriptive  Geometry 8vo, 

Reed's  Topographical  Drawing  and  Sketching 4to, 

Reid's  Course  in  Mechanical  Drawing 8vo, 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design. 8vo, 

Robinson's  Principles  of  Mechanism 8vo, 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo, 

Smith's  Manual  of  Topographical  Drawing.     (McMillan.) 8vo, 

Warren's" Elements  of  Plane  and  Solid  Free-hand  Geometrical  Drawing,  umo, 

Drafting  Instruments  and  Operations i2mo 

Manual  of  Elementary  Projection  Drawing nmo, 

Manual  of  Elementary  Problems  in  the  Linear  Perspective  of  Form  and 

Shadow i2mo, 

Plane  Problems  in  Elementary  Geometry i2mo, 

Primary  Geometry i2mo, 

Elements  of  Descriptive  Geometry,  Shadows,  and  Perspective 8vo, 

General  Problems  of  Shades  and  Shadows 8vo, 

Elements  of  Machine  Construction  and  Drawing 8vo, 

Problems,  Theorems,  and  Examples  in  Descriptive  Geometry 8vo, 

Weisbach's  Kinematics  and  Power  of  Transmission.    (Hermann  and  Klein)8vo, 

Whelpley's  Practical  Instruction  in  the  Ait  of  Letter  Engraving i2mo, 

Wilson's  (H.  M.)  Topographic  Surveying 8vo, 

Wilson's  (V.  T.)  Free-hand  Perspective 8vo, 

Wilson's  (V.  T.)  Free-hand  Lettering 8vo, 

Woolf' s  Elementary  Course  in  Descriptive  Geometry Large  8vo, 


ELECTRICITY  AND   PHYSICS. 

Anthony  and  Brackett's  Text-book  of  Physics.     (Magie.) Small  8vo,  3  00 

Anthony's  Lecture-notes  on  the  Theory  of  Electrical  Measurements.  .  .  .  i2mo,  1  00 

Benjamin's  History  of  Electricity 8vo,  3  00 

Voltaic  Cell 8vo,  3  00 

Classen's  Quantitative  Chemical  Analysis  by  Electrolysis.     (Boltwood.).Svo,  3  00 

Crehore  and  Squier's  Polarizing  Photo-chronograph 8vo,  3  00 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  i6mo,  morocco,  5  00 
Dolezalek's    Theory    of    the    Lead   Accumulator    (Storage    Battery).      (Von 

Ende.) nmo,  2  50 

Duhem's  Thermodynamics  and  Chemistry.     (Burgess.) 8vo,  4  00 

Flather's  Dynamometers,  and  the  Measurement  of  Power mno,  3  00 

Gilbert's  De  Magnete.     (Mottelay.) 8vo,  2  50 

Hanchett's  Alternating  Currents  Explained umo,  1  00 

Hering's  Ready  Reference  Tables  (Conversion  Factors') i6mo,  morocco,  2  50 

Holman's  Precision  of  Measurements 8vo,  2  00 

Telescopic    Mirror-scale  Method,  Adjustments,  and   Tests.  ..  .Large  8vo,  75 

Kinzbrunner's  Testing  of  Continuous-Current  Machines 8vo,  2  00 

Landauer's  Spectrum  Analysis.     (Tingle.  1 8vo,  3  00 

Le  Chatelien's  High-temperature  Measurements.  ( Boudouard — Burgess.)  i2mo,  3  00 

Lob's  Electrolysis  and  Electrosynthesis  of  Organic  Compounds.  (Lorenz.)  i2mo,  1  00 

9 


2 

1,0 

2 

50 

I 

50 

3 

00 

3 

00 

5 

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2 

00 

5 

00 

2 

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3 

00 

3 

00 

2 

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1 

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1 

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3 

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3 

00 

7 

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2 

50 

5 

00 

2 

00 

3 

50 

2 

50 

7 

00 

7 

50 

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50 

6 

oo 

6 

50 

5 

00 

5 

50 

3 

oo 

•*  Lyons's  Treatise  on  Electromagnetic  Phenomena.   Vols.  I.  and  II.  8vo,  each,  6  oo 

*  Michie's  Elements  of  Wave  Motion  Relating  to  Sound  and  Light 8vo,  4  00 

Niaudet's  Elementary  Treatise  on  Electric  Batteries.     (Fishback.) nmo,  2  50 

*  Rosenberg's  Electrical  Engineering.     (Haldane  Gee — Kinzbrunner.).  .  .8vo,  1  50 

Ryan,  Norris,  and  Hoxie's  Electrical  Machinery.     Vol.  1 8vo,  2  50 

Thurston's  Stationary  Steam-engines 8vo,  2  50 

*  Tillman's  Elementary  Lessons  in  Heat 8vo,  1  50 

Tory  and  Pitcher's  Manual  of  Laboratory  Physics Small  8vo,  2  00 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo,  3  00 

LAW. 

*  Davis's  Elements  of  Law 8vo,  2  50 

*  Treatise  on  the  Military  Law  of  United  States 8vo, 

*  Sheep, 

Manual  for  Courts-martial i6mo,  morocco, 

Wait's  Engineering  and  Architectural  Jurisprudence 8vo, 

Sheep, 
Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture  8vo, 

Sheep, 

Law  of  Contracts 8vo, 

Winthrop's  Abridgment  of  Military  Law i2mo,  2  50 

MANUFACTURES. 

Bernadou's  Smokeless  Powder — Nitro-cellulose  and  Theory  of  the  Cellulose 

Molecule i2mo,  2  50 

Bolland's  Iron  Founder i2mo,  2  50 

"  The  Iron  Founder,"  Supplement i2mo,  2  50 

Encyclopedia  of  Founding  and  Dictionary  of  Foundry  Terms  Used  in  the 

Practice  of  Moulding i2mo,  3  00 

Eissler's  Modern  High  Explosives 8vo,  4  00 

Effront's  Enzymes  and  their  Applications.     (Prescott.) 8vo,  3  00 

Fitzgerald's  Boston  Machinist i2mo,  1  00 

Ford's  Boiler  Making  for  Boiler  Makers i8mo,  1  00 

Hopkin's  Oil-chemists'  Handbook 8vo,  3  00 

Keep's  Cast  Iron 8vo,  2  50 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control '•  .Large8vo,  7  50 

Matthe ws's  The  Textile  Fibres 8vo,  3  50 

Metcalf's  Steel.     A  Manual  for  Steel-users i2mo,  2  00 

Metcalfe's  Cost  of  Manufactures— And  the  Administration  of  Workshops. 8vo,  5  00 

Meyer's  Modern  Locomotive  Construction 4to>  I0  °° 

Morse's  Calculations  used  in  Cane-sugar  Factories i6mo,  morocco,  1  50 

*  Reisig's  Guide  to  Piece-dyeing 8vo,  23  00 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vo,  3  00 

Smith's  Press-working  of  Metals 8vo,  3  00 

Spalding's  Hydraulic  Cement i2mo,  2  00 

Spencer's  Handbook  for  Chemists  of  Beet-sugar  Houses.    . .  .  i6mo,  morocco,  3  00 

Handbook  for  Sugar  Manufacturers  and  their  Chemists.  .  i6mo,  morocco,  u  00 

Taylor  and  Thompson's  Treatise  on  Concrete,  Plain  and  Reinforced 8vo,  5  00 

Thurston's  Manual  of  Steam-boilers,  their  Designs,  Construction  and  Opera- 
tion  8vo,  5  00 

*  Walke's  Lectures  on  Explosives 8vo,  4  00 

Ware's  Manufacture  of  Sugar,     fin  press.) 

West's  American  Foundry  Practice i2mo,  2  50 

Moulder's  Text-book i2mof  2  50 

10 


Wolff's  Windmill  as  a  Prime  Mover  8vo,    3 

Wood's  Rustless  Coatings:   Corrosion  and  Electrolysis  of  Iron  and  Steel.   8vo,    4 


MATHEMATICS. 

Baker's  Elliptic  Functions 8vo,  1  50 

*  Bass's  Elements  of  Differential  Calculus umo,  4  jo 

Briggs's  Elements  of  Plane  Analytic  Geometry umo,  i«oo 

Compton's  Manual  of  Logarithmic  Computations i2ir.o,  1   50 

Davis's  Introduction  to  the  Logic  of  Algebra 8vo,  1  50 

*  Dickson's  College  Algebra Large  i2mo,  1   50 

*  Introduction  to  the  Theory  of  Algebraic  Equations Large  i2mo,  1   25 

Emch's  Introduction  to  Projective  Geometry  and  its  Applications 8vo,  2  50 

Halsted's  Elements  of  Geometry 8vo,  1   75 

Elementary  Synthetic  Geometry 8vo,  1  50 

Rational  Geometry i2mo,  1  75 

*  Johnson's  (J.  B.)  Three-place  Logarithmic  Tables:   Vest-pocket  size  paper,  15 

100  copies  for  5  00 

*  Mounted  on  heavy  cardboard,  8  <  10  inches,  25 

10  copies  for  2  00 

Johnson's  (W.  W.)  Elementary  Treatise  on  Differential  Calculus     Smah  8vo,  ■\  00 

Johnson's  (W.  W.)  Elementary  Treatise  on  the  Integral  Calculus. Small  8vo,  1   50 

Johnson's  (W.  W.)  Curve  Tracing  in  Cartesian  Co-ordinates i2mo,  1  00 

Johnson's  (W.  W.)  Treatise  on  Ordinary  and  Partial  Differential  Equations. 

Small  8vo,  3  50 

Johnson's  (W.  W.)  Theory  of  Errors  and  the  Method  of  Least  Squares.  12m  •  ,  1   50 

*  Johnson's  (W.  W.)  Theoretical  Mechanics una,  3  00 

Laplace's  Philosophical  Essay  on  Probabilities.     (Trascott  and  Emory.).  12010,  2  00 

*  Ludlow  and  Bass.     Elements  of  Trigonometry  and  Logarithmic  and  Other 

Tables 8vo,  3  00 

Trigonometry  and  Tables  published  separately Each,  2  00 

*  Ludlow's  Logarithmic  and  Trigonometric  Tables 8vo,  1  00 

Maurer's  Technical  Mechanics 8.    ,  4  00 

Merriman  and  Woodward's  Higher  Mathematics 8vo,  5  00 

Merriman's  Method  of  Least  Squares 8vo,  2  00 

Rice  and  Johnson's  Elementary  Treatise  on  the  Differential  Calculus. .  Sm.  8vo,  3  00 

Differential  and  Integral  Calculus.     2  vols,  in  one Small  8vo,  2  50 

Wood's  Elements  of  Co-ordinate  Geometry 8vo,  2  00 

Trigonometry:  Analytical,  Plane,  and  Spherical 12 mo,  1  00 


MECHANICAL  ENGINEERING. 

MATERIALS  OF  ENGINEERING,  STEAM-ENGINES  AND  BOILERS. 

Bacon's  Forge  Practice nmo,  1  50 

Baldwin's  Steam  Heating  for  Buildings i2mc,  2  50 

Barr's  Kinematics  of  Machinery 8vn,  2  50 

*  Bartlett's  Mechanical  Drawing 8vo,  3  00 

*  "  "  "        Abridged  Ed 8vo,     1   50 

Benjamin's  Wrinkles  and  Recipes i2mo,     2  00 

Carpenter's  Experimental  Engineering 8vo,    6  00 

Heating  and  Ventilating  Buildings 8vo,    4  00 

Cary's  Smoke  Suppression  in  Plants  using  Bituminous  Coal.     (In  Prepara- 
tion.) 

Clerk's  Gas  and  Oil  Engine Small  8vo,    4  00 

Coolidge's  Manual  of  Drawing 8vo,  paper,     1  00 

Coolidge  and  Freeman's  Elements  of  General  Drafting  for  Mechanical  En- 
gineers  Oblong  4to,    2  50 

11 


Cromwell's  Treatise  on  Toothed  Gearing nmo,  I  50 

Treatise  on  Belts  and  Pulleys i2mo,  1  50 

Durley's  Kinematics  of  Machines 8vo,  4  00 

Flather's  Dynamometers  and  the  Measurement  of  Power i2mo,  3  00 

Rope  Driving i2mo,  2  00 

Gill's  Gas  and  Fuel  Analysis  for  Engineers nmo,  1  25 

Hall's  Car  Lubrication nmo,  1  00 

Hering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  morocco,  2  50 

Hutton's  The  Gas  Engine 8vo,  5  00 

Jamison's  Mechanical  Drawing 8vo,  2  50 

Jones's  Machine  Design: 

Part  I.     Kinematics  of  Machinery 8vo,  1  50 

Part  II.     Form,  Strength,  and  Proportions  of  Parts. . 8vo,  3  00 

Kent's  Mechanical  Engineers'  Pocket-book i6mo,  morocco,  5  00 

Kerr's  Power  and  Power  Transmission 8vo,  2  00 

Leonard's  Machine  Shop,  Tools,  and  Methods.     (In  press.). 

Lorenz's  Modern  Refrigerating  Machinery.     (Pope,  Haven,  and  Dean.)     (In  press.) 

MacCord's  Kinematics;   or,  Practical  Mechanism 8vo,  5  00 

Mechanical  Drawing 4to,  4  00 

Velocity  Diagrams 8vo,  1  50 

Mahan's  Industrial  Drawing.     (Thompson.) 8vo,  3  50 

Poole's  Calorific  Power  of  Fuels .8vo,  3  00 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  00 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design. 8vo,  3  00 

Richard's  Compressed  Air nmo,  1  50 

Robinson's  Principles  of  Mechanism •.  .  .  .  8vo,  3  00 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  00 

Smith's  Press-working  of  Metals 8vo,  3  00 

Thurston's   Treatise   on   Friction  and   Lost   Work   in   Machinery   and   Mill 

Work 8vo,  3  00 

Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics .  nmo,  1  00 

Warren's  Elements  of  Machine  Construction  and  Drawing 8vo,  7  50 

Weisbach's    Kinematics    and    the    Power    of    Transmission.     (Herrmann — 

Klein.) 8vo,  5  00 

Machinery  of  Transmission  and  Governors.     (Herrmann — Klein.).  .8vo,  5  00 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  00 

Wood's  Turbines 8vo,  2  50 


MATERIALS   OF    ENGINEERING. 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Burr's  Elasticity  and  Resistance  ot  the  Materials  of  Engineering.     6th  Edition. 

Reset 8vo,  7  50 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Johnson's  Materials  of  Construction 8vo,  6  00 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Martens's  Handbook  on  Testing  Materials.     (Henning.) 8vo,  7  50 

Merriman's  Text-book  on  the  Mechanics  of  M?terials 8vo,  4  00 

Strength  of  Materials nmo,  1  00 

Metcalf 's  Steel.     A  manual  for  Steel-users nmo,  2  00 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vo,  3  00 

Smith's  Materials  of  Machines nmo,  1  00 

Thurston's  Materials  of  Engineering 3  vols.,  8vo,  8  00 

Part  II.     Iron  and  Steel 8vo,  3  5<> 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  so 

Text-book  of  the  Materials  of  Construction 8vo,  5  ot 

12 


Wood's    De  V.)  Treatise  on  the  Resistance  of  Materials  and  an  Appendix  on 

the  Preseivation  of  Timber 8vo, 

Wood's  (De  V.)  Elements  of  Analytical  Mechanics 8vo, 

food's  (M.  P.)  Rustless  Coatings:    Corrosion  and  Electrolysis  of  Iron  and 
SteeL 8vo, 


2  oo 
j  oo 


4  oo 


STEAM-ENGINES  AND  BOILERS. 


Berry's  Temperature-entropy  Diagram i2mo 

Carnot's  Reflections  on  the  Motive  Power  of  Heat     (Thurston.) i2mo 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book. ..  .i6mo,  mor. 

Ford's  Boiler  Making  for  Boiler  Makers i8mo 

Goss's  Locomotive  Sparks 8vo 

Hemenway's  Indicator  Practice  and  Steam-engine  Economy i2mo 

Hutton's  Mechanical  Engineering  of  Power  Plants 8vo 

Heat  and  Heat-engines 8vo 

Kent's  Steam  boiler  Economy 8vo 

Kneass's  Practice  and  Theory  of  the  Injector 8vo 

MacCord's  Slide-valves 8vo 

Meyer's  Modern  Locomotive  Construction 4to 

Peabody's  Manual  of  the  Steam-engine  Indicator i2mo 

Tables  of  the  Properties  of  Saturated  Steam  and  Other  Vapors 8vo 

Thermodynamics  of  the  Steam-engine  and  Other  Heat-engines 8vo 

Valve-gears  for  Steam-engines 8vo 

Peabody  and  Miller's  Steam-boilers 8vo 

Pray's  Twenty  Years  with  the  Indicator Large  8vo 

Pupin's  Thermodynamics  of  Reversible  Cycles  in  Gases  and  Saturated  Vapors 

(Osterberg. ) i2mo 

Reagan's  Locomotives:   Simple   Compound,  and  Electric i2mo 

Rontgen's  Principles  of  Thermodynamics.     (Du  Bois.). 8vo 

Sinclair's  Locomotive  Engine  Running  and  Management i2mo 

Smart's  Handbook  of  Engineering  Laboratory  Practice i2mo 

%>ow's  Steam-boiler  Practice 8vo 

Spangler's  Valve-gears 8vo 

Notes  on  Thermodynamics i2mo 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo 

Thurston's  Handy  Tables 8vo 

Manual  of  the  Steam-engine 2  vols.,  8vo 

Part  I.     History,  Structure,  and  Theory 8vo 

Part  H.     Design,  Construction,  and  Operation 8vo 

Handbook  of  Engine  and  Boiler  Trials,  and  the  Use  of  the  Indicator  and 

the  Prony  Brake 8vo 

Stationary  Steam-engines 8vo 

Steam-boiler  Explosions  in  Theory  and  in  Practice i2mo 

Manual  of  Steam-boilers,  their  Designs,  Construction,  and  Operation 8vo 

Weisbach's  Heat,  Steam,  and  Steam-engines.     (Du  Bois.) 8vo 

Whitham's  Steam-engine  Design 8vo 

Wilson's  Treatise  on  Steam-boilers.     (Flather.) i6mo 

Wood's  Thermodynamics,  Heat  Motors,  and  Refrigerating  Machines. .  .8vo 


25 
50 


2  00 

2  00 

5  00 

5  00 

4  00 

1  50 

2  00 
10  00 

1  50 


5  00 
2  50 


2   50 


: 

^5 

2 

50 

S 

00 

2 

00 

2 

5>J 

3 

00 

2 

50 

I 

00 

i 

00 

1 

50 

0 

OO 

0 

OO 

0 

uo 

5 

00 

2 

50 

1 

50 

5 

00 

5 

00 

MECHANICS  AND  MACHINERY. 


Barr's  Kinematics  of  Machinery 8vo, 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo, 

Chase's  The  Art  of  Pattern-making i2mo, 

Church's  Mechanics  of  Engineering . .  .8vo, 

13 


2  50 
7  50 
2  50 
6  00 


3 

50 

4 

00 

3 

50 

7 

50 

10 

00 

4 

00 

Church's  Notes  and  Examples  in  Mechanics 8vo,  2  oo 

Compton's  First  Lessons  in  Metal-working i2mo,  i  50 

Compton  and  De  Groodt's  The  Speed  Lathe 121x10,  1  50 

Cromwell's  Treatise  on  Toothed  Gearing i2mo,  1  50 

Treatise  on  Belts  and  Pulleys nmo,  1  50 

Dana's  Text-book  of  Elementary  Mechanics  for  Colleges  and  Schools.  .i2mo,  1  50 

Dingey's  Machinery  Pattern  Making i2mo,  2  00 

Dredge's  Record  of   the   Transportation  Exhibits  Building  of  the   World's 

Columbian  Exposition  of  1893 4to  half  morocco,  5  00 

Du  Bois's  Elementary  Principles  of  Mechanics: 

Vol.      I.     Kinematics 8vo, 

Vol.    II.     Statics 8vo, 

Vol.  HI.     Kinetics '. 8vo, 

Mechanics  of  Engineering.     Vol.    I Small  4to, 

Vol.  II '. Small  4to, 

Durley's  Kinematics  of  Machines 8vo, 

Fitzgerald's  Boston  Machinist i6mo,  1  00 

Flather's  Dynamometers,  and  the  Measurement  of  Power nmo,  3  00  . 

Rope  Driving nmo,  2  00 

Goss's  Locomotive  Sparks 8vo,  2  00 

Hall's  Car  Lubrication i2mo,  1  00 

Holly's  Art  of  Saw  Filing i8mo,  75 

James's  Kinematics  of  a  Point  and  the  Rational  Mechanics  of  a  Particle.  Sm.8vo,2  00 

*  Johnson's  (W.  W.)  Theoretical  Mechanics nmo,  3  00 

Johnson's  (L.  J.)  Statics  by  Graphic  and  Algebraic  Methods 8vo,  2  00 

Jones's  Machine  Design: 

Part    I.     Kinematics  of  Machinery 8vo,  1  50 

Part  n.     Form,  Strength,  and  Proportions  of  Parts 8vo,  3  00 

Kerr's  Power  and  Power  Transmission 8vo,  2  00 

Lanza's  Applied  Mechanics 8vo,  7  50 

Leonard's  Machine  Shop,  Tools,  and  Methods.     (In  press.) 

Lorenz's  Modern  Refrigerating  Machinery.      (Pope,  Haven,  and  Dean.)      (In  press.) 

MacCord's  Kinematics;   or,  Practical  Mechanism 8vo,  5  00 

Velocity  Diagrams 8vo,  1  50 

Maurer's  Technical  Mechanics 8vo,  4  00 

Merriman's  Text-book  on  the  Mechanics  of  Materials 8vo,  4  00 

*  Elements  of  Mechanics i2mo,  1  00 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  00 

Reagan's  Locomotives:   Simple,  Compound,  and  Electric nmo,  2  50 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  00 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design. 8vo,  3  00 

Richards's  Compressed  Air nmo,  1  50 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

Ryan,  Norris.  and  Hoxie's  Electrical  Machinery.     Vol.  1 8vo,  2  50 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  00 

Sinclair's  Locomotive-engine  Running  and  Management nmo,  2  00 

Smith's  (0.)  Press-working  of  Metals 8vo,  3  00 

Smith's  (A.  W.)  Materials  of  Machines nmo,  1  00 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo,  3  00 

Thurston's  Treatise  on  Friction  and  Lost  Work  in    Machinery  and    Mill 

Work t 8vo,  3  00 

Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics. 

nmo,  1  00 

Warren's  Elements  of  Machine  Construction  and  Drawing 8vo,  7  50 

Weisbcch's  Kinematics  and  Power  of  Transmission.    (Herrmann — Klein.  ).8vo,  5  00 

Machinery  of  Transmission  and  Governors.      (Herrmann — Klein. ).8vo,  5  00 

Wood's  Elements  of  Analytical  Mechanics 8vo,  3  00 

Principles  of  Elementary  Mechanics nmo,  1  25 

Turbines 8vo.  2  50 

The  World's  Columbian  Exposition  of  1893 4to,  1  00 

14 


METALLURGY. 

t'gleston's  Metallurgy  of  Silver,  Gold,  and  Mercury: 

Vol.    I.     Silver 8vo.  7  50 

Vol.  II.     Gold  and  Mercury 8vo,  7  50 

**  Iles's  Lead-smelting.     (Postage  p  cents  additional.) i2mo,  2  50 

Keep's  Cast  Iron 8vo,  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe 8vo,  1  50 

Le  Chatelier's  High-temperature  MeasuremePts.  (  Boudouard — Burgess,  inmo,  3  00 

Metcalf's  Steel.     A  Manual  for  Steel-users     i2mo,  2  00 

Smith's  Materials  of  Machines 121110,  1  00 

Thurston's  Materials  of  Engineering.     In  Three  Parts 8vo,  8  00 

Part    II.     Iron  and  Steel 8vo ,  3  50 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo,  3  00 

MINERALOGY. 

Barringer's  Description  of  Minerals  of  Commercial  Value.    Oblong,  morocco,  2  50 

Boyd's  Resources  of  Southwest  Virginia 8vo  3  00 

Map  of  Southwest  Virignia Pocket-book  form.  2  00 

Brush's  Manual  of  Determinative  Mineralogy.     (Penfi>ld.) 8vo,  4  00 

Chester's  Catalogue  of  Minerals 8vo,  paper,  1  00 

Cloth,  1   25 

Dictionary  of  the  Names  of  Minerals 8vo,  3  50 

Dana's  System  of  Mineralogy Large  8vo,  half  leather,  12  50 

First  Appendix  to  Dana's  New  "  System  of  Mineralogy." Large  8vo,  1   00 

Text-book  of  Mineralogy 8vo,  4  00 

Minerals  and  How  to  Study  Them i2mo,  1  50 

Catalogue  of  American  Localities  of  Minerals Large  8vo,  1  00 

Manual  of  Mineralogy  and  Petrography i2mo  2  00 

Douglas's  Untechnical  Addresses  on  Technical  Subjects i2mo,  1  00 

Eakle's  Mineral  Tables 8vo,  1   25 

Egleston's  Catalogue  of  Minerals  and  Synonyms 8vo,  2  50 

Hussak's  The  Determination  of  Rock-forming  Minerals.    (Smith. ). Small  8vo,  2  00 

Merrill's  Non-metallic  Minerals.   Their  Occurrence  and  Uses 8vo,  4  00 

*  P&nfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo  paper,  o  50 
Roseabusch's    Microscopical   Physiography   of   the    Rock-making  Minera  s 

(Iddings.> 8vo.  5  00 

*  Tillman's  Text-book  of  Important  Minerals  and  Rocks 8vo.  2  00 

Willi&*ns's  Manual  of  Lithology 8vo,  3  00 

MINING. 

Beard's  Ventilation  of  Mines l2mo.  2  50 

Boyd's  Resources  of  Southwest  Virginia 8vo.  3  00 

Mtp  of  Southwest  Virginia Pocket  book  form.  2  00 

Douglas's  Untechnical  Addresses  on  Technical  Subjects i2mo.  1  00 

*  Dririer's  Tunneling,  Explosive  Compounds,  and  Rock  Drills    .4to.hf  mor  25  00 

Eissltr'6  Modern  High  Explosives 8vo  4  00 

Fowler's  Sewage  Works  Analyses .  .12010  2  00 

Goodyear's  Coal-mines  of  the  Western  Coast  of  the  United  States i2mo.  2  50 

Ihlseng's  Manual  of  Mining 8vo.  5  00 

**  Iles's  Lead-smelting.     (Postage  oc  additional.) i2mo.  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe 8vo.  1   50 

O'DriscoU's  Notes  on  the  Treatment  of  Gold  Ores 8vo.  2  00 

*  Walke's  Lectures  on  Explosives 8vo,  400 

Wilson's  Cyanide  Processes i2mo,  1   50 

Chlcrtoation  Process iamo,  1  50 

15 


Wilson's  HydrauLv  axel  .Placer  Mining i2mo,  2  00 

Treatise  on  Fractkal  and  Theoretical  Mine  Ventilation. nmo,  1  25 

SANITARY  SCIENCE. 

Folwell's  Sewerage.     (Designing,  Construction,  and  Maintenance.) 8vo,  3  oc 

Water-supply  Engineering 8vo,  4  00 

Fuertes's  Water  and  Public  Health i2mo,  1  50 

Water-filtration  Works nmo,  2  50 

Gerhard's  Guide  to  Sanitary  House-inspection i6mo,  1  00 

Goodrich's  Economic  Disposal  of  Town's  Refuse Demy  8vo,  3  50 

Hazen's  Filtration  of  Public  Water-supplies 8vo,  3  00 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control 8vo,  7  50 

Mason's  Water-supply.  (Considered  principally  from  a  Sanitary  Standpoint)  8vo,  4  00 

Examination  of  Water.     (Chemical  and  Bacteriological.) nmo,  1  2$ 

Merriman's  Elements  of  Sanitary  Engineering 8vo,  2  00 

Ogden's  Sewer  Design i2mo,  2  00 

Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Refer- 
ence to  Sanitary  Water  Analysis i2mo,  1  25 

*  Price's  Handbook  on  Sanitation i2mo,  1  50 

Richards's  Cost  of  Food.     A  Study  in  Dietaries nmo,  1  00 

Cost  of  Living  as  Modified  by  Sanitaiy  Science i2mo,  1  00 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sanitary  Stand- 
point  8vo,  2  00 

*  Richards  and  Williams's  The  Dietary  Computer 8vo,  1  50 

Rideal's  Sewage  and  Bacterial  Purification  of  Sewage 8vo,  3  50 

Tumeaure  and  Russell's  Public  Water-supplies 8vo,  5  00 

Von  Behring's  Suppression  of  Tuberculosis.     (Bolduan.) i2mo,  1  00 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

Woodhull's  Notes  on  Military  Hygiene 16m©,  1  50 

MISCELLANEOUS. 

De  Fursac's  Manual  of  Psychiatry.  (Rosanoff  and  Collins.).  .  .  .Large  i2mo,  2  50 
Emmons's  Geological  Guide-book  of  the  Rocky  Mountain  Excursion  of  the 

Interr.ational  Congress  of  Geologists Large  8vo,  1  50 

Ferrel's  Popular  Treatise  on  the  Winds 8vo.  4  00 

Haines's  American  Railway  Management nmo,  2  50 

Mott's  Composition,  Digestibility,  and  Nutritive  Value  of  Food.  Mounted  chart,  1  25 

Fallacy  of  the  Present  Theory  of  Sound i6mo,  1  00 

Ricketts's  History  of  Rensselaer  Polytechnic  Institute,  1824-1894.  .Small  8vo,  3  00 

Rostoski's  Serum  Diagnosis.     (Bolduan.) nmo,  1  00 

Rotherham's  Emphasized  New  Testament Large  8vo,  2  00 

Steel's  Treatise  on  the  Diseases  of  the  Dog 8vo,  3  50 

Totten's  Important  Question  in  Metrology 8vo,  2  50 

The  World's  Columbian  Fxposition  of  1893 4to,  1  00 

Von  Behring's  Suppression  of  Tuberculosis.     (Bolduan.) nmo,  1  00 

Winslow's  Elements  of  Applied  Microscopy nmo,  1  50 

Worcester  and  Atkinson.     Small  Hospitals,  Establishment  and  Maintenance; 

Suggestions  for  Hospital  Architecture :  Plans  for  Small  Hospital  .nmo,  1  25 

HEBREW  AND  CHALDEE  TEXT-BOOKS. 

Green's  Elementary  Hebrew  Grammar nmo,  1  25 

Hebrew  Chrestomathy 8vo,  2  00 

Gesenius's  Hebrew  and  Chaldee  Lexicon  tr    the  Old  Testament  Scriptures. 

(Tregelles.) Small  4to,  half  morocco,  5  00 

Lettews's  Hebrew  Bible 8vo>  2  25 

.6 


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COLUMBIA  UNIVERSITY  LIBRARIES      I 

This  book  is  due  on  the  date  indicated  below,  or  at  the     1 
expiration  of  a  definite  period  after  the  date  of  borrowing,  as     1 
provided  by  the  rules  of  the  Library  or  by  special  arrange-     1 
ment  with  the  Librarian  in  charge.                                                 J 

DATE  BORROWED 

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DATE  BORROWED 

DATE  DUE                j 

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| 

QP514 
Hemmarst 


H18 
1904 


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